CA2111962C - Amplification of target nucleic acids using gap filling ligase chain reaction - Google Patents
Amplification of target nucleic acids using gap filling ligase chain reaction Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6862—Ligase chain reaction [LCR]
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
An improved "gap filling" embodiment of the Ligase Chain Reaction (LCR) is described. Gap filling LCR is LCR
wherein at least one of the probes is recessed so that a gap is formed between the adjacent probes when they are hybridized to target.
The gap is filled using polymerase and deoxyribonucleotide triphosphates before ligation of the probes together. There are single and double gap versions, depending on whether one or two probes are recessed and require filling before ligation. The improve-ment resides in selecting and using target sequences such that only a single type, or two types, of deoxyribonucleotide triphos-phate(s) are required to fill double gaps each being 1-10 bases in length, preferably 1-3 bases. Probes having specific sequences are claimed for a number of pathogens.
wherein at least one of the probes is recessed so that a gap is formed between the adjacent probes when they are hybridized to target.
The gap is filled using polymerase and deoxyribonucleotide triphosphates before ligation of the probes together. There are single and double gap versions, depending on whether one or two probes are recessed and require filling before ligation. The improve-ment resides in selecting and using target sequences such that only a single type, or two types, of deoxyribonucleotide triphos-phate(s) are required to fill double gaps each being 1-10 bases in length, preferably 1-3 bases. Probes having specific sequences are claimed for a number of pathogens.
Description
AMPLIFICATION OF TARGET NUCLEIC ACIDS
USING GAP FILLING LIGASE CHAIN REACTION
BACKGROUND
This invention relates to a method of performing ligase chain reaction (LCR) amplification and, particularly, to a method of ligase chain reaction amplification wherein at least one of the probes is reversibly modified at the intended point of ligation so that it is not a substrate for the ligase catalyzed reaction.
Exemplary modifications include the absence of one or more nucleic acid bases to form a "recess".
The modified end prevents or reduces target independent blunt-end ligation of probe 1 S duplexes and is later corrected in a target dependent manner to enable ligation.
In many cases, the feasibility of a nucleic acid based diagnostic assay is dependent an the ability to amplify the signal generated by only a few molecules of target. Although signal amplification is one potential solution, target amplification is often the preferred solution in nucleic acid based assays. Target amplification involves the repeated copying or duplication of sections of the nucleic acid designated as the target.
In the target amplification technique known as polymerase chain reaction (PCR ~ a pair of primers are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The pr~rners are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to increase geometrically the number of target sequence molecules.
PCR is described further in U.S. Patents 4,683,195 and 4,683,202.
An alternate mechanism for target amplification is known as ligase chain reaction (LCR). In LCR, two primary (first and second probes) and two secondar~~
(third a,~d fourth) probes are employed in excess. The first probe hybridizes to first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or iigate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a WO 93/OOA47 ~ ~ ~ ~ ~ ~) ~ . PC'1f/US92/O5~'_'' fourth (secondary) probe cah hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance.
Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. In order to understand LCR and the improvements described herein, it is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A-320 308 and EP-A-439 182.
A potential problem associated with ligase chatn reaction is background signal caused by target independent ligatian of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These t 5 duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent blunt-end ligation of these duplexes is a relatively rare event, it is sufficiently common: to cause undesirably high background signals in diagnostic assays.
Some attempts to overcome this background problem have been published. Far example, 4V0 90/01069 (Segev Diagnos>:ics) and GB 2 225 1 12 A tlmperial Chemical industries Plc.> describe versions of a ligation-based amplification scheme which inciudes~a palymerase-mediated gap-fiiliryg step prior to ligation. In addition, EP-A-439 l82 teaches variations of LCR that reduce background. One such variation involves gap filling and lig~tion. However, these references teach nothing about selection of the particular target sequences as taught and claimed in the present apPl iGat i an.
it is therefore a primary object of the present invention to improve the sensitivity of nucleic acid based assays by decreasing the occurrence of target independent ligation which causes falsely positive background signal. This object is met in the present invention by modifying at least one probe end so that when hybridized with its complementary probe, the resulting duplex is not "blunt-ended"
ti.e. ligatable) with~respect to the partner complementary probe duplexes.
After hyaridizing to the target, the modified ends are "corrected" in a target dependent fashion to render the adjacent probesligatable. Several features of the probes and the associated target sequences taught in this application make this task particularly elegant . .
USING GAP FILLING LIGASE CHAIN REACTION
BACKGROUND
This invention relates to a method of performing ligase chain reaction (LCR) amplification and, particularly, to a method of ligase chain reaction amplification wherein at least one of the probes is reversibly modified at the intended point of ligation so that it is not a substrate for the ligase catalyzed reaction.
Exemplary modifications include the absence of one or more nucleic acid bases to form a "recess".
The modified end prevents or reduces target independent blunt-end ligation of probe 1 S duplexes and is later corrected in a target dependent manner to enable ligation.
In many cases, the feasibility of a nucleic acid based diagnostic assay is dependent an the ability to amplify the signal generated by only a few molecules of target. Although signal amplification is one potential solution, target amplification is often the preferred solution in nucleic acid based assays. Target amplification involves the repeated copying or duplication of sections of the nucleic acid designated as the target.
In the target amplification technique known as polymerase chain reaction (PCR ~ a pair of primers are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The pr~rners are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to increase geometrically the number of target sequence molecules.
PCR is described further in U.S. Patents 4,683,195 and 4,683,202.
An alternate mechanism for target amplification is known as ligase chain reaction (LCR). In LCR, two primary (first and second probes) and two secondar~~
(third a,~d fourth) probes are employed in excess. The first probe hybridizes to first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or iigate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a WO 93/OOA47 ~ ~ ~ ~ ~ ~) ~ . PC'1f/US92/O5~'_'' fourth (secondary) probe cah hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance.
Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. In order to understand LCR and the improvements described herein, it is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A-320 308 and EP-A-439 182.
A potential problem associated with ligase chatn reaction is background signal caused by target independent ligatian of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These t 5 duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent blunt-end ligation of these duplexes is a relatively rare event, it is sufficiently common: to cause undesirably high background signals in diagnostic assays.
Some attempts to overcome this background problem have been published. Far example, 4V0 90/01069 (Segev Diagnos>:ics) and GB 2 225 1 12 A tlmperial Chemical industries Plc.> describe versions of a ligation-based amplification scheme which inciudes~a palymerase-mediated gap-fiiliryg step prior to ligation. In addition, EP-A-439 l82 teaches variations of LCR that reduce background. One such variation involves gap filling and lig~tion. However, these references teach nothing about selection of the particular target sequences as taught and claimed in the present apPl iGat i an.
it is therefore a primary object of the present invention to improve the sensitivity of nucleic acid based assays by decreasing the occurrence of target independent ligation which causes falsely positive background signal. This object is met in the present invention by modifying at least one probe end so that when hybridized with its complementary probe, the resulting duplex is not "blunt-ended"
ti.e. ligatable) with~respect to the partner complementary probe duplexes.
After hyaridizing to the target, the modified ends are "corrected" in a target dependent fashion to render the adjacent probesligatable. Several features of the probes and the associated target sequences taught in this application make this task particularly elegant . .
According to one feature, the probes have recesses relative to the point of ligation which create a gap when hybridized to the target. The gap is then filled in a target dependent manner to render the probes ligatabte. Gap filling can be accomplished by extension of one or more probes, preferably two probes.
Targets are selected and probes are designed so that only one, or a maximum of two, of the four deoxyribonucleotide triphosphates is needed to fill both the gaps.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a method for detecting the presence of a target nucleic acid sequence in a sample, said method employing the ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target sequence, said method comprising:
a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula:
S'-(N)hXEp.FqZ(N)k-3' wherein E represents any base, F represents any base except E, p and q are independently integers from 1 to about 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k> each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected;
b) providing a plurality of each of four probes having the formulas:
A 5'-(N)h~ X Ep-3' A': 3'-(N')htX'-5' B: 5'-Z (N)k~-3' B': ~ 3'-F'qZ'(N')k~-5' wherein the symbols have the same meanings given above, except that h' and k' need only approximate h and k, respectively, varying by no more than 25~, and wherein at least one of the probes is labeled with a moiety capable of detection; and also providing deoxyribonucleotide triphosphates of E' and F, a polymerase reagent and a ligase reagent;
c) performing the following cycle at least once:
i> mixing said probes with said sample under hybridizing Conditions to allow probes to hybridize to the target sequence and'its complement if present, or to reorganized probes created therefrom;
i i ) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F
', ~cr~u~9~~o~ ._., Wo 93~dDO447 deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerise reagent try adding E' deoxyribonucleotide triphosphates to Its 3' end iii»igating extended probe A to probe B, and extended probe B' to probe A', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
d) separating reorganized probe molecules from unreorganized labeled probes; and e) detecting the presence of said label in the reorganized or fraction as a measure of the presence of the target sequence.
In especially preferred embodiments, F is E' so that both gaps can be filled by the deoxyribonucleotide triphosphate of only E'. Preferably p and q are small, ie., between 1 and 3, inclusive. Integers p and q may be equal ar may differ try one or more. Preferably, the label comprises one or more haptens covalently bound to at 1 S least one of the probes.
In another aspect, the invention relates to diagnostic kits for the detection of a target nucleic acid sequence in a sample, said nucleic acid having a target sequence comprising 5'-(N)hXEp.FqZ(N)k-3' wherein E and F independently represent any base, p and q are independently integers from 1 to about 10, X represents any erase except E or F', Z represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p*h> and (q*k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic far the target sought to be detected, said kit comprising in combination:
(a) four probes, having the formulas:
A: 5'-f N)ht X Ep-3, A': 3'-( N' )I,~X'-5' B: 5~-Z (N>~~-3, B'; 3'-F'qZ'tN')ky-5' wherein the symbols have the same meanings given above, except that h' and k' need only approximate h and k, respectively, varying by no more than 25~, and wherein at least one of the probes is labeled with a moiety capable of detection;
(b) a polymerise reagent capable of extending probe A with said polymerise ~5 reagent in a target dependent mahner by adding F deoxyribonucleotide triphosphates to its 3' end; and extending probe B' with said polymerise reagent in a target dependent manner by adding E' deoxyribonucleotide triphosphates to its 3' end, thereby to render the primary probes iigatable to one another when hybridized to target and, ~.. :.. , : . ,. .; <;.. . .~,;;.. . :;: . ,, , ; , ,; :,: .., .;, .,.,.
vV~ 93!00447 ~ ~ ~ ~ ~ ~ ~ P~('/US921054'77 optionally, to render the secondary probes ligatable to one another when hybridized to target;
(c> deoxyribonucteotide triphosphates of E' and F; and td) a ligase reagent for ligating the extended probe A to probe B, and extended probe S' to probe A', thereby to form reorganized probe molecules.
Especially preferred kits, also include means for separating reorganized probe molecules from unreorganized labeied probes and/or means for detecting the detectable label.
In yet another aspect, the invention relates to compositions of matter each comprising a mixture of four probes, the four probes being selected from specific . sets of sequences given in the examples. These compositions of matter have utility in detecting the presence of certain pathogens according to the methods described above.
It will, of course, be realized that sequences being slightiy shorter or longer than the ~, ~ ~ exemplified and claimed sequences are deemed to fall within the scope of the 1 S invention, provided they are capable of hybridizing with the same locations on the targets as the claimed sequences.
EiRIEF DESCRIPTION OF THE DRAWINGS
Figure t is a graphic representation of the process of ligase chain reaction as ~0 it is known in the prior art.
Figure 2 is a graphic representation of a generalized, double gap variation of the invention.
Figure 3 is a graphic representation of a somewhat more specific double gap variation of the invention.
W~J 93J~0447 '~ ~ i .,~' .,. .- ~' PCf/US'~2/O5~'"7 DETAILED DESCRIRTlON
For purposes of this invention, the target sequence is described to be single stranded. However, this should be understood to include the ease where the target is actually double stranded but is simply separated from its complement prior to hybridization with the probes. In the case of double stranded target, the third and fourth (secondary) probes, A' and B', respectively, will participate in the initial step by hybridizing to the target complement. In the case of single stranded target, they will not participate in the initial hybridization step, but wail participate in subsequent hybridization steps, combining with the primary fused sequence produced by ligating the first and second probes. Target sequences may comprise deoxyribonucleic acid (DNA> or ribonucleic acid (RNA).
Target sequences may be the nucleic acid of virtually any entity which contains nucleic acid. For example, but not limitation, the bacteria and viruses ~, r ~ illustrated in the examples have nucleic acid target sequences. Target sequences may, but need not, represent pathogenic organisms or viruses. Target sequences may also represent nucleic acid which is being examined for the presence of a specific allele, or for a specific mutation of deletion, such as is often the case in genetic testing.
Finally; target sequences may be simply any nucleic acid sequence of general interest, such as may be the case in forensic analysis of DNA.
Throughout this application, the "prime" ('> designation is used to indicate a complementary base,or sequence, A probe is "complementary" to another probe if it hybridizes to the other probe and has substantially complementary base pairs in the hybridized region, Thus, probe A can be complementary to A' even though it may have ends not coterminal with A': The same is true of B and B'. Similarly, the short sequences Xn and Ym have complementary sequences designated a~ X'n and Y'm, respectively. Finally, the complement of a single base, e.g. Q, is designated as Q'. As used herein with respect to sequences;''complementary" encompasses sequences that have mismatched base pairs in the hybridizable region, provided they can be made to hybridize under assay conditiohs.
It is also to be understood that the term "the 4 bases" shall refer to Guanine (G), Cytosine tC), Adenine (A) and Thymine (T) when the context is that of DNA; beat shall refer to Guanine (G), Cytosine (C), Adenine (A) and Uracil (U) in the context of RNA. The term also includes analogs and derivatives of the bases named above.
Although the degenerate base lnosine (I) maybe employed with this invention, it is not preferred to use l within modified portions of the probes according to the invention.
It is an impørtant feature of the present invention that instead of using two pairs of probes capable of forming blunt-ended duplexes, at least one probe of one of the probe pairs initially includes a "modified" end which renders the resultant duplex "nonblunt" and/or not a suitable substrate for the ligase catalyzed fusion of the two probe duplexes. A "modified end" is defined with respect to the point of ligation rather than with respect to its complementary probe. A "modified end" has omitted bases to create a "gap" between one probe terminus and the next probe terminus (See, e.g., probes A' and B, of Figs. 2 and 3:) By convention in this application, a modified end is referred to herein as a "recess", the recess being the gap between two primary or secondary probes after hybridizing to the target. The presence of these modified ends reduces the falsely positive signal created by blunt-end ligation of complementary probe duplexes to one another in the absence of target.
"Correction" of the modification is subsequently carried out to render the probes ligatable. As used herein "correction" refers to the process of rendering, in a target dependent manner, the two primary probes or the two secondary probes ligatable to their partners. Thus, only those probes hybridized to target, target complement or polynucleotide sequences generated therefrom are "corrected."
"Correction" can be accomplished by several procedures, depending on the type of modified end used.
As used herein, "point of ligation" or "intended point of ligation" refers to a specific location between two probe partners that are to be ligated in a template-dependent manner. It is the site at which the "corrected" probe lies adjacent its partner in 3' hydroxyl-S' phosphate relationship. For each set of four LCR
probes there are two "points of ligation", a point for the primary probe partners and a point for the secondary probe partners. In conventional LCR the two points of ligation are opposite one another, thus forming blunt ended duplexes when the probe pairs hybridize to one another. In the present invention, the points of ligation are not opposite one another; but are displaced from one another in the "recess"
embodiments by one or more bases by virtue of the gaps. The exact point(s> of l;igation varies depending on the embodiment and, thus, this term is further defined in the context of each embodiment.
Each of the probes may comprise deoxyribonucleic acid (DNA) or ribonucleic a~~'~i (RNA). It is a routine matter to synthesize the desired probes using c ventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, CA); DuPont, (Wilmington, DE); or Milligen, (Bedford, MA>. Phosphorylation of the S' ends of the appropriate probes (eg. A' and B), while necessary for ligation by ligase, may be accomplished by a kinase or by commercial synthesis reagents, as is known in the art.
* Trade-mark ~rV~ 93/40~i447 ; r . ~ . ' , PC~'/US~2/O5a''~
'~'.~~~~(~~
Throughout this application, the bases X, Y and Q, and their complements are described as being selected from certain subsets (P! or M> of the 4 bases. In reality, the sequences are not "selected" at all, but are dictated by the sequence of the target strand. The term "selected" in this context is taken to mean that a target sequence having the desired characteristics is located and probes are constructed around an appropriate segments) of the target sequence, In general, the methods of the invention comprise repeated steps of (a) hybridizing the modified probes to the target (and, 1f double stranded so.that target complement is present, to the target complement>; (b) correcting the modification in i 0 a target dependent manner to render the probes ligatable; (c) ligating the corrected probe to its partner to form a fused or ligated product; and (d> dissociating the fused product from the target and repeating the hybridization, correction and ligation steps to amplify the desired target sequence. Steps (a), (c) and (d) are essentially the same for all of the embodiments and can be discussed together. They are generally the same steps that one would employ in conventional LCR. Step (b) varies depending on the type of modification employed and each different type is discussed separately.
Hybridization of probes to target (and optionally to target complement) is adequately explained in the prior art; e.g EP-320 308. Probe length, probe concentration and stringehcy of conditions all affect the degree and rate at which hybridization will occur. Preferably, the probes are sufficiently long to provide the desired specificity; i.e, to avoid being hybridizable to nontarget sequences in the sample. Typically, probes on the order of 15 to t 00 bases serve this purpose.
Presently preferred are probes having a length of from about 1 S to about 40 bases.
The probes are added in approximately equimolar concentration since they are expected to react stoichiometrically. Each probe is present in a concentration ranging from about S nanomolar (nM) to about 90 nM; preferably from about 10 nM to about nM. For a standard reaction volume of 50~L, this is equivalent to adding from about 3 x 10 t t to about 1 x 10 ~ 2 molecules of each probe; and around S
x,10 ~ ~
molecules per 50 uL has been a good starting point. The optimum quantity of probe 30 used for each reaction also varies depending on the number of cycles which must be performed and, of course, the reaction volume. Probe concentrations can readily be determined by one of ordinary skill i~ this art to provide optimum signal for a given number of cycles.
The stringency of conditions is generally known to those in the art to be 35 dependent on temperature; solvent and other parameters. Perhaps the most easily controlled of these parameters is temperature and thus it is generally the stringency parameter varied in the performance of LCR. Since the stringency conditions required for practicing this invention are not unlike those of ordinary LCR, further '~~'O ~3/00~7 ~ ~ ~ ~ P(_'T/iJS92/05~77 _g_ detail is deemed unnecessary, the routine practitioner being guided by the examples which follow.
The next step in the general method follows the specific correction step and comprises the ligation of one probe to its adjacent partner. Thus, each corrected primary probe is ligated to its associated primary probe and each corrected secondary probe is ligated to its associated secondary probe. An "adjacent" probe is either one of two probes hybridizable with the target in a contiguous orientation, one of which lies with its phosphorylated 5' end in abutment with the 3' hydroxyl end of the partner .
probe. "Adjacent" probes are created upon correction of the modified ends) in a target dependent manner. Since enzymatic tigation is the preferred method of covalently attaching two adjacent probes, the term "ligation" wail be used throughout the application. However, "ligation" is a general term and is to be understood to include any method of covalently attaching two probes. ~ne alternative to enzymatic ligation is photo-ligation as described in EP-A-324 616.
The conditions and reagents which make possible the preferred enzymatic ligation step are generally known to those of ordinary skill in the art and are disclosed in the references mentioned in background. Ligating reagents useful in the present invention include T4 ligase; and prokaryotic ligases such as E coli iigase, and Thermus thermophi?us ligase (e.g., ATCC 27634) as taught in ~P-320 308. This latter ligase is presently preferred for its ability to maintain activity during the thermal cycling of LCR: Absent a thermal ~y stable ligase, the ligase must be added again each time the cycle is repeated. Also useful are eukaryotic ligases, including DNA lipase of Drosophilia; reported by Robin, et al., ,J. Bfol. Chem.
261:10637-10647 ( 1986 >. .
Once ligated, the fused; reorganized probe is dissociated (e.g. melted) from the targe and, as with conventional LCR; the process is repeated for several cycles. The number of repeat cycr ~s may vary from fo about 100, although from about 15 to about 70 are preferred presently. , (t is desirable to design probes so that when hybridized to their complementary (secondary) probes, tho ends away from the point of intended ligation are not able themselves to participate in other unwanted ligation reactions.
Thus, li~atable sticky or blunt ends should be avoided. If such ends must be used, then S' terminal phosphates should be avoided, eliminated w blocked. This can be accomplished either through synthesizing oligonucleotide probes (which normally carry no 5' terminal phosphate groups), or through the use of phosphatase enzymes to remove terminal phosphates (e.g, from oligonucleotides generated through restriction digests of DNA). Alternatively, ligation of the "wrong" outside ends of the probes can be prevented by blocking the end of at least one of the probes with a "hook"
~ ~ ~. ~ 9 ~ 2 ~~~U~~z~~s~~~
-lo-or marker moiety as will be described in detail below. In the absence of one of the above techniques, the outside ends of the probes can be staggered so that if they are joined, they will not serve as template for exponential amplification.
Following amplification, the amplified sequences can be detected by a number of conventional ways known in the art. Typically, detection is performed after separation, by detemining the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction. Separation may be accomplished by electrophoresis, by chromatography or by the preferred method described below.
!n a particularly preferred configuration, haptens, or "hooks"', are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of ail four probes. A °'hook" is any moiety having a ', p . specific ligand-receptor affinity. Typically, the hooks) at one end of the fused product (e.g. the 5' end of A and the 3' end of A') comprises an antigen or hapten capable of being immobilized by a specific binding reagent (such as antibody or avidin> coated onto a solid phase. The hooks) at the other end te.g. th@ 3' end of B and the S' end of B') contains a different antigen or hapten capable of being recognized by a label or a label system such as an antibody-enzyme conjugate. Exemplary hooks include biotin, fluorescein and digoxin among many others known in the art. A
substrate is then added which is converted by the enzyme to a detectable product.
EP-A-330 221 to Enzo describes oligonucleotides having a biotin molecule attached at one end.
E't'!p~MODIF~ED BY ~ -ESSE
In this embodiment; modified ends are created by eliminating from one or yore of the probes'a short sequence of bases, thereby leaving a recess or gap between the 5' end of one probe and the 3' end of the other probe when they are both hybridized to the target for target complement, or polynucleotide generafied therefrom).
In order for LCR to amplify the target, the gaps between the probes must be filled in f i.e., the modification must be "corrected"). In a first version, this can be done using a polymerase or a reverse transcriptase and an excess of deoxyribonucleotide triphosphates which are complementary to the target strand apposite the gap.
However, prior to,discussing this embodiment in detail, a brief digression on set terminology may be helpful: A set te.g., S> consists of alt the elements contained within the set S: The set "not S" then consists of all the remaining elements of the "Universe" which are not found in S. The "tJhiverse" for purposes of this application consists of the four bases G, C, A and T, ar G, C; A and U as described above.
The . Y.:. .. 4 ~'..1: n . , ..,...... . ,...,-..;,.. . .. ..,...,... . ::?~' .....z'::~. . .,~..e::
......,:;::.. .....~....., ., .:~':...:~.....,.oY. .. tu. ;:.:'. . 'a::~'i .
....r.'.::., n. ..~:,' ..,. ; .::: ,. ,. ~,~:..
intersection of set S and another set (e.g., R) consists only of those elements which are found in both S and R. Thus, as used in this application, the set "not N
and not M"
consists of those bases which are present in neither the gap Xn nor the gap Ym.
According to this invention, the set "not N and not M" must not be an empty set; i.e at least one base must remain in this set to code for the "stopbase".
Gap Filling by Extension:
In accordance with this first version, the invention involves repeated steps of (a> hybridizing the probes to the target (and, if double stranded so that target complement is present, to the target complement); (b> extending at least one probe to fill in at least one gap, designated Xn; (c) llgating the extended probe to the adjacent probe to form a fused or ligated product; and (d> dissociating the fused product from the target and repeating the hybridization, extension and ligation steps to amplify the desired target sequence.
In this version, which includes both single gap ("SG"> and double gap ("DG") configurations, the "gaps" Xn and Ym which impart the "modified ends" are "corrected" by extending one or both of the modified probes using a polymerase or a reverse transcriptase. Generally, extension of a probe hybridized to a DNA
target is accomplished by a DNA polymerase or a Klenow fragment as is known in the art.
In the case of an RNA target, extension is accomplished t a reverse transcriptase.
Exemplary reverse transcriptases include those from avian myeloblastosis virus (AMV) and Moioney murine leukemia virus (M-MuLV) generally available to those skilled in the art. Certain DNA polymerases will also recognize RNA as template under certain conditions. It is, of course, preferable to utilize extension reagents which are thermally stable and can withstand the cycling of high temperatures required for LCR. If the extension reagent is not thermally stable, it typically must be re-added at each cycle of 1.CR. Such thermostable polymerases~presently include AmpliTaqTM, (available from Cetus-Perkin Eimer), Thermos polymerase (available from Molecular Biology Resources,lnc. Milwaukee. WI, "MBR") and recombinant or purified native polymerases from Thermos aQuaticus, Thermos thermophilus or other species known to be thermostable.
Correction by extension in this manner requires the presence in the reaction mixture of deoxyribonucleotide triphosphates (dNTP's) complementary to the bases of the target in the gap region(s). More specifically, for a gap having the sequence Xn, the dNTP's that must be supplied are designated dX'TP wherein X' stands for the the complements of each base in the gap X~. The dNTP's are commercially available from a number of sources, including Pharmacia (Piscataway, ttJ) and Bethesda Research Laboratories (Gaithersburg, MD>.
* Trade-mark ~r~ ~3ioo~7 . . . ~crms~zios~~7 Extension must be terminated precisely at the point of ligation so that the extended probe abuts'the adjacent probe and can be ligated to it. "Stopbases"
are employed for this purpose, (See figure 2). A "stopbase", designated D', is defined in terms of its complement, Q and is accomplished by omitting from the reaction S mixture, dNTP's that are complementary to Q; i.e. by omitting dQ'TP from the reaction mixture. Thus it is seen how the bases for the gap sequence(s> must be selected from a set, N, consisting of only three of the four bases, so that the complementary three of the four dNTP's are added to the reaction mixture. When the fourth dNTP, dQ'TP, is absent from the reaction mixture extension will terminate at the desired point of ligation. It follows that Q' is the first base in the adjacent probe, and the base on the target which codes for the stopbase is the first base adjacent the gap (on the S' end of the X~ gap in Figure 2).
White the concept is easiest to grasp in the SG configuration, it should be understood that the SG variation is merely a special case of the double gap (DG) 1 S variation discussed below (in SG, m=0). However, only the DG configuration is relevant to the presently claimed invention. As shown in Fig 2, a first probe, A, hybridizes to a first segment of the target strand, T. A second probe, B, hybridizes to a ~ecohd segment pf: the target strand, leaving a gap of one or more bases between the two probes This gap is designated Xn on the target. Following extension and ligation, a third ps~obe, A', is hybridizable to a first portion of reorganized probe, A:B; and a fourth probe; B', is hyoridizabie to a second portion of reorganized probe, A:B. In the DG configuration, the: third probe, A', and the fourth probe, B'; hybridize such that a gyp of one or more bases lies between the probes. This gap is designated Y,~
on the reorganized probe (and on target comptemeht). As is shown in Fig. 2, the target 2S strand, T, may be double stranded, having a complement, T'. In this case, the third and fourth probes may participate in the initial hybridization by hybridizing to first and second segments'of the-target Complement.
Extension by polymerase or transcriptase proceeds in a 5' to 3' direction.
Consequently, the 3' ends of both A and 8' will be extendable by polymerase in the absence of anything to prevent extension. Extension is terminated when the next base called for by the template is absent from the reaction mixture. Thus, probe A
is extended through gap Xn until stopbase complement (Q> is encountered along the target trahd. Similarly, probe B' is extended through gap Ym until stopbase complement (Q> is encountered (either on the target complement or on the A
half of ~5 reorganized A:B>. Neither probe A' nor B will serve as a template for extension of A
Qr B'; so probes A and B' are extended only if hybridized to the target (or to reorganized polynucleotide products from previous cycles).
t. ., ° ' . ;.,. . - . ,.. ,; ;. . :. , . ~ ;
WO 93/00447 ~ ~ ~ ~ ~ PC'~'/US92/05477 As alluded to above, it is important to terminate the extension of 'A and B' at the end of the respective gaps (i.e., at the point of ligation) so that the extended probe can be iigated to the 5' end of the adjacent probes, B and A'. Therefore, the reaction mixture omits the deoxyribonucleotide triphosphate complementary to the base (a>
S immediately adjacent the S' end of gaps Xn, and Ym. Of course, it will be understood that it is not required that the same base stop extension in both directions.
A different base can be used provided it is not needed to fill either of the gaps. it should now be apparent that the actual points of ligation in this embodiment are always at the S' ends of probes A' and B. It is not by mere coincidence that these are also the locations of the stopbases Q'.
Accordingly, the gaps Xn and Ym can be any number of bases long, i.e., n can be any integer greater than or equal to 1, and m is any integer greater than 0.
It is to realized, however, that the choice of which gap is Xn and which is Ym is arbitrary in the first place; but n and m cannot both be zero. The gaps need not be the same length, ~,r' i.e., m need not equal n. When, m equals zero, the double gap variation degenerates into the specialized case of the single gap, which is not used in the embodiment being claimed herein. Tho only restriction on the bases X is that they be selected from a Set N which consists of from 1 to any 3 of the four bases. Similarly, the bases Y
are drawn from set M. Since at least one stopbase Q' must be maintained, the combined sets N and M which represent the possible bases for X and Y, respectively, must include no more than three of the four bases. Accordingly, Y can be from zero to any three of the four bases provided that at least one base remains in the set "not N and not M". If set N cor(stitutes less than three of the four bases, then Y can be a base that is not within N so long as there is at least one base remaining, the complement of which can serve as the stopbase Q' for termination of probe extension. A single stopbase can serve to terminate extension ih both the Xn and Ym gaps.
A second limitation on sequence Ym occurs if m equals n. If the gaps are the same length, the sequence Ym should not be complementary to the sequence Xn or the 3' ends of probes A and,B' would constitute "sticky ends". "Sticky ends" would permit a target independent double stranded complex to f orm wherein probe A
hybridizes to probe B' such that'ligations and amplification would proceed. Rather, when m equals n it is preferred that Ym not be complementary to X~. In other words, the ends of probes A and B' shauld at least be "slippery ends" which may be the same length, but are not complementary.
in a preferred aspect of the invention, the fourth probe B' includes a 3' terminal sequence of X~, identical in length to the Xn sequence gap in the target. This arrangement is not essential to the invention, however, as the gap need only be formed between the probes. Thus, the 3' terminus of the fourth probe B' may stop short of W~U 93I004~t7 ~ ~ ~ ~ ~ ~ ~ PCT/UB92/05~"'7 the 3' end of sequence Xn, provided there is no 3' recessed end with respect to the second probe B. Since extension occurs in a S' to 3'' direction and dX'TPs must be present anyway (to extend through Xn), probe B' would be extended through the gap, (bath Ym and any remainder of Xn > just as the first probe A is extended through the Xn gap.
The general method of the invention employing the double gap embodiment is now described briefly. First, probes A, and B are allowed to hybridize to target, if present. If the target is double stranded, it is first denatured and probes A' and B' can also hybridize to the target complement. In the presence of a suitable polymerase, probes A and B' are extended by the addition of dX'TPs and dY'TPs, respectively, to their 3' ends using the target strand as a template. ~iowever, when the polymerase encounters the base Q on the template, it is unable further to extend probe A
since dQ'TP is not provided in the reaction mixture as dt~TP's. Extension terminates precisely at the point of ligation with the extended 3' end of probe A
abutting the 5' end of probe B. It is not known if the presence of probes A' and B on the template will terminate extension. But even in their absence, extension is terminated if attention is paid to proper stopbase selection.
Next, a ligase is employed to join the 3' hydroxyl end of extended probe A to the S' phosphate end of probe B to form a double stranded complex of fused or ligated primary probe and target strand. If the target is double stranded and has a complement T', the ligase will also join probes A' and B' in the initial cycle if they are hybridized to the target complement. If they are hybridized to excess probes A
and B
rather than target complement, ligation is inhibited since the ends are neither blunt nor sticky and there is no substrate for ligation.
Subsequently, the double stranded complexes are dissociated and new probes A, A', B and B' are permitted to hybridize to the target, the target complement, and both of the fused polynucleotides from the first cycle. Extension and ligation occur as befpre and the process can be repeated.
Some exemplary combinations of X's and Y's, their dNTP counterparts and the resultant possibilities for 0 and Q' are given in Table I.
..:..,_.... . ..,.:.... ., ,.
'VV~ 93/OD447 ~ ~ ~ ~ PCf/U~92/05A77 _1S-TABLE I
ILLUSTRATIVE GAP SEQUENCES, REDUIRED dNTPs, and POSSIBLE
COMBINATIONS FOR 0 and Q' IN DOUBLE GAP VARIATION
~n,L.C! YmLM X'TPs Y'TPs j.'~t and ~TO..P~AsE
A A T T T,C,G A,G,C
G 'f C A C, A G, T
AT AT T, A T,A C, G C, G
,r.,C C,e, T, G C,T T A
ATG AAA T, A, C T C G
G G C AAACG ~, G T,G,C T A
C
ATTGA AGGT T, A, C T,C,A C G
~ ~ G G C G complement.
C
not permitted The set not N,~ not M provides the possible complements (Q) for the stopbase D'.
The actual stopbase (Q') possibilities are given in the next column.
In tine case of double gap s, the length of gaps Xn and Ym may be one or any integer greater than one. For example, gyps of from t to 20 bases would be possible.
Practically, however, gaps of much shorter length are preferred; for example from 1 to 3 or S bases: It has been found that gaps of just one or two bases greatly increase the ratio of true signal t:o background and leave the largest number of options for stopbases and dXTP's. Since probes are actually designed around existing targets, 1 S rather than "selecting' stopbases, single or two base gaps are desirable in most cases.
Furthermore, it has been discovered that the double gap embodiment is preferred to the single gap version.
TARGET SEQUENCE CHARACTERlSTlCS
For clarity; double gaps are represented herein as "DG p,q" where "p" is the number of bases in the gap in one strand, and "4" is the number of bases in the gap of the other strand. Thus, d preferred double gap embodiment has two bases missing from each of the two probes whose 5' end participates in the ligation, and is designated DG 2,2. in this preferred embodiment, the 3° ends of the other two probes 25 do not overlap; rather they terminate at the same point on the target strand (and its complement). Also for clarity in this application the use of a dot or period "."
V1~~'l 9100447 PCT/~JS~2/05~~7 2~.~.~(.~~
between bases in a target sequence represents the point at which the probes, when hybridized to their complementary probes, would be blunt-ended but for the gaps in two of the probes. In other words, probes A and B' (see Fig. 3> have 3' termini which, absent the gaps in probes A' and B, would be blunt-ended at the point of , ligation. It is this point at which ligation would have occurred that is designated by a dot. However, with gaps present according to the invention, probes A and B' are extended, thus creating two, offset points of actual ligation.
In a particularly preferred DG embodiment bath the gaps are finable with just one type of deoxyribonucleotide triphosphate. This requires selecting a target sequence having the sequence -Ep.E'q-, where E represents any base, and p and q are integers between 1 and about 10. The probe sets must be designed so that the two probes whose ~' ends participate in the extension/ligatton (eg. probes A and B' tn Fig.
3) both terminate at the dot "." between E and E'. In order to provide a "stopbase" a further requirement is added. The target sequence must have a base other than E in 1 S the next position outward from the E string; and, similarly, must have a base other than E' in the next position outward from the E' string. This can be represented as -LEp.E'qJ-, where L is any base other than E and J is any base other than E'.
Final ly, to provide sufficient probe length for easy hybridization and, optionally, to provide specificity; the regions (N>h and (N)k are employed, to give:
5'-( N)hLEp.E'qJ(N)k-3' where N represents any base independently of any other base, N; and h and k are each integers greater than 5. Generally, to provide the desired specificity, the individual probes must be at least 10 or 1 1 nucleotides long, i.e., the sums (p*h> and (q*k) must be at least 10; more usually will be about 20 to 40, but can be up to 100 or more. As mentioned, p and q may be the same or different, and generally will be small, eg. 1-3.
In this preferred DC 2,2 system, probes would be designed and synthesized as follows; using the designations given in Fig. 3:
A' Hapten~--3'_(N')r,~L~_5' 3'-Eq.J'(N'>k~-5~°Hapten~ B' A Hapten~_5'_(N)h, L Eh_3' s'-J (N)~,t-5~-Hapten2 B
wherein the terms are defined as above except that h' and k' need not equal h and k exactly. It is entirely permissitrle for any of the probes to be slightly shorter or lohger than either the target sequehce or the complementary probe. This variation in length is indicated by the ""' symbol. The variation is usual ly no more tnan lu-25% of the probe length; preferably 10~ or less. Of course, no variation in length is also possible. Moreover, hand k' may represent a different number at each WO 9/00447 ~ ~ ~ ~ PCI'~US92/0~47~
occurrence, provided they are within the 20- 25~ variation limit; thus, probe A
need not be exactly p bases langer than probe A' (their (N)ht portions may wary in length too) and the same is true for k' and probes B and B'.
The single deoxyribonucleotide triphosphate of E' only is needed to fill all the S gaps. The bases L and J perform the same function as Q, i.e. to code for the stopbase Q'. However, designations L and J are used here because of the additional restrictions placed on them in this embodiment. As described elsewhere in this specification, haptens 1 and 2 serve to enable separation and detection of ligated products.
They are particularly useful for automated detection in the IMxo instrument (Abbott Laboratories). Napten 1 should be different than hapten 2. Virtually any low molecular weight compound to which antibodies can be raised may serve as hapten.
Several examples of this preferred embodiment are given in the first column of Table II below and in the Examples which fallow.
Another more generalized embodiment of the invention utilizes target sequences and probe sets wherein just two types.of deoxyribonucleotlde triphosphates are needed to fill all the gaps. Examples of this embodiment are given in the second ' column of Table I I and its target sequence is represented by the fallow~ng formula:
5"°(N)hXEp.FaZ(N)k-3, wherein N, p, q, h and k are all defined as before. E may be any base as before. F may be any base except E (if it were E the ends would be "sticky" when p=q), but when it is E' this case degenerates to the simpler embodiment described ,above.
Because of the additional flexibility in the choice of F, additional restrictions are placed on the stop bases X and Z. X may be any base but E or F', while Z may be any base but F or E'.
The gaps are filled by he deoxyribonucleatide triphosphates of E' and F.
z5 Ln this DG 2,2 system, probes would be designed ahd synthesized as follows, using the designations given ire Fig. 3:
A' Hapted~_3'_(N~)r~~~_5' 3'_F,aZ~(N'>~.t--5~-Napten2 B
A Hapteni_s'_(N)ht X Ep_3' y_Z (N)k9-s~~-Hapten2 B
The two deoxyribonucleotide triphosphates needed to fill all the gaps are F
and E'. The bases X and Z perform the same function as Q to cads for the stop base Q'.
However, designations X and Z are usbd here because of the additional restrictions placed on ahem in this embodiment. the restrictions are ~tso different than the restrictions on 35 L and J. The haptens are as described above:
Ct is important the "X" which codesfor a stopbase here (or X or X' used in a probe) should not be confused with the Xn gap or the dX'Tps needed to fail the Xn gap.
Regrettably, there are only twenty-six letters in the alphabet and most have been '~O 93/00447 PCTlUS92/05~''~
used in this application. It is believed that confusion between the two uses of X and X' is avoided by the context.
Other variations of this invention include DG configurations other than 2,2.
As shown in Table f I and in the examples, DG 1, t ; DG 1,2 (as well as 2, t ); DG 2,3 S (as well as 3,2> and DG 3,3 are also possible. In addition, gaps of up to 10 bases are included in the generic p and q case, although Table I I I below shows the low probability of gap junctions longer than about 3. 'There may be some advantage to having p be approximately equal to q (i.e. varying by only 1 or 2), but this is not a strict requirement. Though not tested, there is no reason to believe a double gap of 1,3 or 2,4 will not also work in the invention.
The above discussion describes in detail the "Special " symbois used in Table II. The "Conventional" symbols will not need further explanation to those of ordinary skill in the art.
TABLE II
~t 5 EXEMPLARY SINGLE and TWO dNTP FILL
DOUBLE GAP ("DG") JUNCTIONS
(The dot "."serves only to align sequences in the Table and to divide between right and left probe sets: All targets are written with their 5' end to the left. ) Gaps finable with just one Gaps fillable with just two deoxyribonucleotide deoxyribonucleotide triphosphate type triphosphates types - ~N)hLE.E'J(N)k (N>hXE,FZ(N)k DG 1,1 Generic; E' fills eneric cases)E' & F fill generic cases) (in all,~ (in all Subgeneric: (N)r,DC.GH(N)kG fills (N)hKC.TM(N)kG&T fill (N)r,HG.GD(N>kC fills (N>hYG.TR(N)kC&T fill (N>r,BA.TV(N)kT fills (N>r,RC.AY(N>kG&A fiil (N)hVT.AB(N)kA fills (N>hMG.AK(N)~C&A fill V~'~ 93100487 . ~ 1'C°if/~JS92/05477 DG 1,2,and 2,1 (N>hLE.E'E'JtN)k (N>hXE.FFZ(N>k Subgeneric: (N)hDC.GGH(N)k G fills(N)hKC.TTM(N)k G&T fill (N)hl-iG.CCDtN)k C tN)hKCC.TMtN)k G&T fill fills (N)hBA.TTV(N)k T fills(N)hYG.TTR(N)k C8~T fill tN)hVT.AAB(N)k A fills(N)hYGG.TR(N)~ CST fill (N)hRC.AAY(N)k G&A fill .
tN)hRCC.AY(N)k G&A fill (N)hMG.AAKtN)~ C&A fill (N)hMGG,AK(N)k C&A fill DG 2 2 Generic: tN)hLEE.E'E'JCN)k (N>hXEE.FFZtN)k Subgeneric: (N>hDCC.GGt-ltN)k G tN)hKCC.TTMtN)kG&T till fills (N)hHGG.CCDtN)k C fills(N)hYGG.TTR(N)kC&T fill , ~.:' tN)hBAA.TTVtN)k T fillstN)hRCC.AAYtN)kG&A fill tN)hVTT.AAB(N)~ A fillstN)hMGG.AAK(N>kC&A fill DG 2 3 and 3 2 (N)hLEE.E'E'E'J(N)k tN>hXEE.FFFZtN)k SubgeneriC: tN>hDCCC.GGH(N>k G (N)hKCCC.TTM(N)kGB~T fill fills (N>hHGGG.CCDtN)k C (N)hKCC.TTTM(N)kG&T fill fills tN)hBAAA.TTV(N>k T (N)hYGGG.TTR(N)kC8~T fill fills tN>hVTTT.AABtN)k A (N>hYGG.TTTR(N)kC&T fill fills (N)hRCCC.AAYtN)kG&A fill (N)hRCC.AAAYtN)kG&A fill (N>hMGGG.AAK(N)kC&A fill (N)r,MGG.AAAK(N)kC&A fill Generic: (N)hLEEE.E'E'E'JtN)k (N)hXEEE.FFFZ(N)k DG 3,~
__ (N)r,DCCC.GGGH(N)k (N)hKCCC.TTTM(N)~, Subgeneric: G fills G&T ffll (N)hHGGG.CCCD(N)~ C (N)hYGGG.TTTR(N)k fills C&T fill tN>~,BAAA.TTTV(N)k (N)nRCCC.AAAY(N)~;
T fills G&A fill tN)hVTTT.AAABtN>k A (N)hMGGG.AAAK(N)k fills C&A fill DG p,a (N)r,LEp.E'qJtN)k (N)hXEp.FaZtN)k GENERAL CASE E' alone fills E' and F alone fill Vl~~'~3/~dD~t7 ~ : : P(.'T/US~32/05~"''~
where the symbols have the following meanings:
CONVENTIONAL SPECIAL
A Adenine E any base .
B any base. but not A) F any base adenine C Cytosine L any base except E
D any base but cytosin(not J any base except E' C) G Guanine X any base except E or F' H any base but guaninenot G> Z any base except F or E' K G or T!U only h, any integer greater than i k M A or C only p, any integer between 1 and q N any base about 10 R A or G only S C or G only T Thymine U Uracil V any base but thymine/uracil w A or T/U only Y C or T/U onlv It will be understood that those of ordinary skill in the art will know haw to search for and identify specific target sequences meeting the requirements outlined above. For example many databases contain sequence information (eg, GENBANK, NBRF, EMBL or Swiss-Prot) which can be searched by readily available computer software (eg. MacVector, MacMolly or Geneworks>. It will also be realized that in any organism's known genome, multiple locations meeting the requirements will generally be found. For example, searching just the SF2CG isolate of HIV t (see Examples 1-S>, which contains approximately 9.7 kiiobases, reveals over 2000 possible DG locatiohs. The breakdown for each type of gap and fill situation is given in Table III. It should be realized that when p=q, a DG p,q on one strand at one location also comprises a DG p,q site an the opposite strand. In the situation where F=E' (ie. a single dNTP fill) the two target sites ace really just one location. Thus, for all 1 S symmetric double gaps twhere p~q) the Table reports the number actually found ' by searching. In contrast; an asymmetric DG (eg, a DG 1,2 or DG 2,3) on one strand represents a second, different site (but DG 2;1 or DG 3,2, this time) on the opposite strand. Thus, asymmetric sites are reported in the Table without halving the number.
W~ 93!00447 ~ ~ 1 ~'~ ~ l PC,')f/1<JS92/OS477 TABLE I l l Representative Numbers of Gaps by type in an Organism's Genomic DNA
Gaps finable with just Gaps finable with just Type of GAP one deoxyri;bo- two deoxyriba-nucleotlde nucleotide triphosphate type triphosphates types H1V t (SF 2CG Isotate)1 (9.7 kb) pG 1 1 1053 1099 e. .tr~act~~rn~t~s MOMP2 (2.2 kt?) ~ p . pG i 2 120 136 _____._._______ _ _. ._..__ 24 ........................_.... i 8 . ____~~_ ~.2 _._,-....,........
C. trachomat~s cryptic plastriid3 (7.5 kb) DG 1 2 4 i 5 488 pG 2.~,...~.-.... 61 55 aG 2 3 39 24 DG 3,3 6 6 , t: Sanchez-Pescado; R., et a1. Science 22'7:484-492 ( 1985), see examples t-5.
2. Baehr, W: et at: Proc.
Natl. Acad. Sct. USA 85:4000-4004 ( 1988) (EMBL Genbank Data Base: lnteiltgenetics, Accession ~J038131, see examples 1 1-12.
3_ Matt, C. et al. Nub. Acids Res. 16:4053-4067 (1988), see example 13.
For the HIV 1 isolate, thirteen of the 75 DG 2,2 single dNTP
fill sites are located within the gag region. These 13 locations awe listed in Appendix A.
In cases where genamic sequences are not known, at least certain portions must be established prior to using any version of LCR.
Typically, one of ordinary 15 skid can determine a sequence through routine methods, such as cloning and sequencing as taught in conventional textbooks on the subject, such as Maniatis, T, et ai. 'Molecular Cloning: A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor; NY (1982 and 1989>.
Whether one is seeking a universal probe set for several types, strains, or 20 serovars, etc. (as might be the case for HIV or Chlamydia?
ar one is seeking a f . _ , ._ ,:
'W~ 93100447 P~1'lt.3S92/05~~7 specific probe set to identify just one type (as might be the case for HPV), one of ordinary skill will know how to compare sequences obtained from such a search with the genomes of other organisms to identify conserved or unique sequences, respectively. In addition, routine laboratory investigations may be needed to confirm the universality or specificity of a particular probe set. The length and nature of tN)h and (N)k may greatly affect the universality or specificity of the respective probes, although it is also possible to design probes such that specificity is gained by the gap regions.
E~a~~~h~~ '.ores In a variation of either "recessed" embodiment, the deoxyribonucleotide triphosphates used in filling the gaps may be modified to contain a marker moiety.
Exemplary markers include direct labels such as radioisotopes, or hooks such as biotin, digoxin or other hapten capable of being recognized by either a solid phase or a 1 S label producing system. Isotopic labels include 32P, and 3H among others.
Incorporation of the marker into the dNTP's is generally a matter of conventional organic chemistry: Linkers or spacers may be used but are not essential. It is only important that the modified dNTP be able to be incorporated into the gap opposite its complement on the target strand and be covalently bonded to the adjacent base.
The invention will now be described further by way of examples which are illustrative of the invention and are not intended to limit it in any way. For example, sequences of specific length are listed. ft should be understood that sequences covering the same map positions but having slightly fewer or greater numbers of bases are deemed to be equivalents of these sequences and fall within the scope of the invention, provided they V~ill hybridize to the same positions on the target as the listed sequences.
Unless otherwise indicated, sequences in the examples are written with the 5' end toward the left. In the examples, some probes are labeled with haptens:
'°FL"
represents a fluorescein hapten and "BIO" represents a biotin hapten, covalently attached to the probe. The haptens are attached at either the 3' or 5' terminus as shown. Throughout the examples, the numbering system for probes corresponds to a map position followed by a decimal .i, .2, .3, or .4, which correspond to probes A, A', B, B', respectively, of Fig. 3. Thus, arranging the probes as: .1 : .3 .2 : .4 W~ 9310(D4~47 ~ ~ ~ ~ ~ ~ ~ P~'1'/1JS92/(D~477 yields an arrangement which is upside-down when compared to Fig. 2 or 3. The map position given is for the left-most base when the probes are aligned as if hybridized.
The remaining map positions can be calculated from this information, knowing that the left-most position bears the lowest number.
In most of the examples, results were read in an IMxo instrument. This is commericaily available from Abbott Laboratories and is described in EP-A-288 and ~in Fiore, M. et al Clin. Chem., 34/9:1726-1732 ( 1988). It should be noted that the IMx instrument typically generates Nmachine" noise or background in the range of 2-8 counts/sec/sec.
Quantities of polymerise are expressed in units, defined (eg., by MBR) as follows: 1 unit of enzyme equals the amount of enzyme required to incorporate nanomoles total nucleotides into acid-insoluble material in 30 min at 70'C.
lj:nits of ligase enzyme are defined (internally by Abbott Laboratories) as: t mg of 9S~
,, ~. ~ purified Thermus thermvphilus DNA ligase has a specific activity of about 1 x 1 Oa t S units. While this is not precisely standardized and may vary by as much as 20~, optimization is wi~hin the skill of the routine practitioner.
Examples 1-12 relate to detection of Human Immunodeficiency Virus (HIV1) , using several different sequences. The sequences, their map position (SF2CG
isolate according to Sanchez-Pescado, R., et al. Science 227:481-~i92 ( 1985)), and their Sequence ID Nos. are given in Table IV, below. Appendix A gives the number and map positions of DG2,2 targets in the gag region of HIV 1.
Table I V.
1667. t : FL-AACCCTTTAG AGACTATGTA GACC
1667.2: TCTACATAGT CTCTAAAGGG TTC-FL 2 1667.3: TTCTATAAAA CTCTAAGAGC CGA-BIO 3 1667.4: BID-CGGCTCTTAG AGTTTTATAG AACC 4 912.1: FL-AGAACGATTC GCAGTCAATC S
CTGG
912.2: AGGATTGACT GCGAATCGTT CTA-FL6 3S 912.3: TGTTAGAAAC ATCAGAAGGC TGC-BIO7 912.4: B10-CAGCCTTCTG ATGTTTCTAA 8 CAGG
2086. t : FL-GCTAATTTTT TAGGGAAGAT 9 CTGG
2086:2: AGATCTTCCC TAAAAAATTA GCC-FL10 2086.3: TTCCTACAAG GGAACGCCAG GGA-Bf01 2086.4: B10-CCCTGGCGTT CCCTTGTAGG i MoD ~93/~~447 ~ ~ ~ ~ ~ ~ ~ PCT/~J~9210~~'''7 789. t : FL-GATGGGTGCG AGAGCGTCGG TATT t 3 789.2: TACCGACGCT CTCGCACCCATCT-FL 14 789.3: GCGGGGGAGA ATTAGATAAA TGG-BIO 15 789.4: B10-CATTTATCTA ATTCTCCCCC GCTT 16 S
t5 508,1: FL-ACCCACTGCT TAAGCCTCAA TAAAG 17 ..
508.2: T1'TATTGAGG CTTAAGCAGT GGGTT-FL 18 508,3: TTGCCTTGAG TGCTTCAAGT AGTGT-BIO t 9 508.4: BIO-CACTACTTGA AGCACTCAAG GCAAG 20 , 5569. t : FL-GAACAAGCCC CAGAAGACCA AGGG ?_ 1 5569.2: TTGGTCTTCT GGGGCTTGTT C-FL 22 5569.3: ACAGAGGGAG CCATACAATG AA-BIO 23 5569.4: BIO-TTCATTGTAT GGCTCCC'TCT GTGG 24 t 450, t : FL--GCATGCAGGG CCTATTGCAC 25 CAGE
1450.2: TGGTGCAATA GGCCCTGCAT G-FL 26 t 450.3: AAATGAGAGA ACCAAGGGGA AG-BIO27 t 450.4: B10-ACTTCCCCTT GGTTCTC'TCA 28 TTTGG
t 5?3. t : FL-AGAAATCTAT AAAAGATGGA 29 THAT
t 573.2: TTATCCATCT.TTTATAGATT TCT-FL30 t 573.3: TGGGATfAAATAAAATAGTAAG-BIO 31 t 573.4: B10-CTTACTATTT T~TTTAATCC 32 CAGG
4060.1: FL-GCATTAGGAA TCATTCAAGC 33 ACAA
4060.2: GTGCTTGAAT GATTCCTAAT GC-FL 34 4060.3: AGATAAGAGT GAATCAGAGT TA-Bi035 4060:4: BIO-TAAC-fCTGAT TCACTCTTAT 36 CTGG
2683. t : FL-AAGGAAGGGA AAATTTCAAA 37 AATT
2683.2: TTTTTGAAAT TTTCCCTTCC TT-FL 38 2683.3: CCTGAAAA'TC CATACAATAC T-B1039 2683.4: Bi0-AGTATTGTAT GGATTTTCAG 40 GCCC
1753. t : FL--AACCTTGTTG GTCCAAAATG 4 CAAA t 1753.2: GCATTTTGGA CCAACAAGGT: f-FL 42 1753.3: AGATTGTAAG ACTATTTTAA A-BIO 43 t 753.4: Bl0-TTTAAAATAG TCTTACAATC 44 TGGG
3697. t : FL-GTAATATGGG GAAAGACTCC'TAAA45 3697.2: AGGAGTCTTT CCCCATATTA C-FL 46 3697.3: AAACTACCCATACAAAAGGAA-BI0 47 3697.4: B10-TTCCTTTTGT ATGGGTAGTT 48 TAAA
Double gap LCR (DG2,2) was performed for 40 cycles consisting of a 65 second incubation at 85°C and a 65 second incubation at 50°C. Reactions were set up with either 0 or t 02 target molecules. The target DNA was a BamH 1 fragment of HI
V t DNA
cloned into pBR322. Negative reactions contained t 0 nanograms of human placental DNA. Rositive reactions contained the indicated number of copies of HI V 1 plasmid DNA
in a background of t 0 nanograms of placental DNA. The LCR oligonucleotides used are listed in Table IV. These oligonucleotides are specific for the map positions t667-woo 9~roo~~ ~ ~ ~ ~ t~°~.~:. ~creus92rosa7~
t 716 within the gag region of the HIV1 genome (HIV SF2CG isolate, see Sanchez-Pescado>. Reactions were run in a buffer containing SOmM EPPS pH 7.8, 100 mM
KCI, 10 mM MgCl2, t mM DTT, 10 mM NH4C1, 100 uM NAD, 10 ug/ml BSA, 5 x 101 1 molecules each oligonucleotides 1667.1, 1667.2, 1667.3, 1667.4 (ID Nos.
S 1-4, respectively>, 1 uM 2'-deoxyguanosine S'-triphosphate, 0.5 units Thermos DNA polymerase (Molecular Biology Resources, Inc., "MBR">, and 3400 units Thermos thsrmophilus DNA ligase. Reaction volume Was SO pl and each reaction was overlaid with 25 ut of mineral oil prior to cycling.
Following amplification, reactions were diluted t:t with IMx di)uent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Num~r of ar,~t molecules $
0 S.?6 102 1686.8 Double gap LCR (DG2,2) was performed for 40 cycles using the same cycling parameters described in Example 1. Reactions were set up in duplicate with either 0, 10 or 102 target DNA molecules. Negative reactions contained 1 microgram of human placental DNA. Positive reactions contained 102 HIV target DNA molecules in 1 microgram of human placental DNA. The oligonucleotides used are listed ~n r ame ~v.
These oligonucleotfd~s are specific for map positions 9 i 2-961 within the gag region of the H(V 1 genome (HIV SF2CG Isolate>. Reactions were run in a buffer containing 50 mM EPPS pH7.8, 20 mM K*; 30 mM MgCl2, 7.5 x 101 1 molecules each oligonucleotides 912.1, 912.2;912.3,912.4 (lD Nos. 5-8, respectively), 100 pM
NAD, 1 pM 2'-deoxycytidine 5'-triphosphate, 0.5 units of Thermos DNA
polymerase (MBR), and 3400, units Therrrus th~rmophilus DNA ligase. Reaction volume was ~l and each reaction was overlaid with 25 t~l of mineral oil.
Following amplification; reactions were diluted 1:1 with IMx diluent buffer and amplification products were detected via a sandwich immunoassay performed with the Abbott IMx automated immunoa~sa~ system.
N~ b . r ~~t 1'0~~
0 3.8 1 Q 32.8 102 604.4 W(J 93/0~447 ~. PCT/US92/0. 7 Double gap LCR (DG2,2) Was performed for 40 cycles consisting of a 65 second incubation at 85°C and a 65 second incubation at 60'C. Reactions were set up in quadruplicate with either 0 or 102 HIV 1 target DNA molecules. Negative reactions contained 1 microgram of human placental DNA. Positive reactions contained 102 HIV
1 target DNA molecules in l microgram of human placental DNA. The ollgonucteotides used are listed in Table IV. These oligonucleotides are specific far map positions 2086-2135 within the gag region of the HIV 1 genome tHIV SF2CG isolate) except that the nucleotide in position 15 of oligonucleotide 20863 was changed from G
to C
to disrupt a possible intramolecular hairpin structure tposition 8 in oligonucteotide 2086.4 changed from C to G). Reactions were run in a buffer identical to that used in example 2 except that oligonucleotides 2086.1, 2086.2, 2086.3, and 2086.4 tID
Nos. 9-12, respectively) were used at 7.5 x 101 1 mol,ecutes each per reaction.
Following amplification; r~eactians were diluted 1:1 with IMx dituent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~i~llb~t of ar~et m_.plecules 0 5.9 , 02 249.5 Exai lie 4.
Doable gap LCR tDG2;2) was performed for 40 cycles using cycling parameters identical to those used in Example 3. Reactions were set up quadruplicate with either O or 102 HIV l target DNA rnolecules. Negative reactions contained 1 microgram of human placental DNA, Positive reactions contained 102 HIV 1 target DNA
molecules in 1 microgram of human placental DNA. The oligonucleotides used are listed in Table IV. These oligonucleotidesare'specific far map position 789-838 within the gag region of'the HI V 1 gen~me tHl V SF2CG isolate?. Reaction conditions were identical to those in Example 2 except that oligohuc)eofides 789.1, 789.2, 789.3, and 789.4 at 7.5 x 101 1 each ( ID Nos. l 3- t 6, respectively), and 2-deoxyadenosine 5'-triphosphate at 1 ~M were used, Following amplification, reactions were diluted i:i with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
j~,~ber of tart molecules $~rg 0 3.8 p2 604.4 W~ 93/~D044? ~ ~ ~ ~ ~ ~ PCT/US92/0547?
Double gap LCR tDGl,1> was performed for 45 cycles consisting of a 6S second incubation at 85°C and a 65 second incubation at 65'C. Reactions were set up in duplicate with~either 0 or 102 target DNA molecules. Negative reactions contained nanograms of human placental DNA. Positive reactions contained 102 HIV 1 target DNA molecules in 10 nanograms of human placental DNA. The oligonucleotides used are specific for map posttions 508-559 within the LTR region of the HIV 1 genome ' fHIV SF2CG isolate). Reactions were run in a buffer identical to that used in Example 10 1 except that oligonucleotides 508.1, 508.2, 508.3, and 508.4 tID Nos. 17-20, respectively) were used at 5 x i 011 molecules each and 2'-deoxycytidine S'-triphosphate was used at 1 uM.
Following amplification, reactfions were diluted 1:1 with IMx diiuent buffer, and the LCR amplification products wire detected v1a a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
L~,"~'~l<L ~~C9~.~olecules 0 4.5 102 1284.8 ~amnle 6.
Double gap LCR (DG2,3) is performed far 30-SO cycles as in the previous examples. The oiigonucleotides used are listed in Table IV, and are specific for map positions 5569-5516 within the vpu coding region of the HI V 1 genome (HI V
isolate): Reaction conditions are like those described in Examples 1 - 3, except that approximately 5 x 101 1 molecules each of otigonucleotides 5569.1, 5569.2, 5569.3, 5569.4 (ID Nos. 21-24; respectively, and approximately 1 p.M 2'-deoxycytadine S'-triphosphate are used.
Following amplification, the reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
JE,~ ~!~ l e?.
Double gap LCR (DG2,2) was performed for 4S cycles consisting of a 6S second incubation at 90°C and a 65 second incubation at 65'C. Reactions were set up in duplicate with either O or 102 HIV 1 target DNA molecules. Negative reactions contained 10 nanograms of human placental DNA. Positive reactions contained HIV 1 target molecules in 10 nanograms of placental DNA. The oligonucleotides used are listed in Table IV. These oligonucleotides are specific for map positions 1498 within the gag region of the HIV 1 genome (HIV SF2CG isolate). Reactions were run in a buffer identical to that described in Example 2 except that S x 101 1 molecules each of oligonucleotides 1450.1, 1450.2, 1450.3, and 1450.4 (ID Nos.
S 25-28, respectively) were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number of target molecules , Rate 0 4.8 102 1932.5 Double gap LCR (DG2,1: 2 dNTPs> is performed for 30-50 cycles as in the previous examples. The oligonucleotides used are listed in Table IV, and are specific for map positions 1573-1620 within the gag region of the HIV 1 genome tHIV
isolate). Reaction conditions are similar to those described in Examples 1 -3, except that approximately 5 x 101 1 molecules each of oligonucleotides 1573.1, 1573.2, 1573.3, 1573.4 (ID Nos. 29-32, respectively), and approximately 1 uM each 2'-deoxycytidine S'-triphosphate and 2'-deoxyadenosine 5'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Double gap LCR (DG2,2: 2 dNTPs) are performed for 30-50 cycles as in the previous examples. The oligonucleotides used are Listed in Table IV, and are specific for map positions 4060-4107 within the poi region of the HIV i genome (HIV
isolate). Reaction conditions are similar to those described in Examples 1 -3, except that approximately S x 101 1 molecules each of oligonucteotides 4060.1, 4060.2, 4060.3, 4060.4, LID Nos. 33-36, respectively) and approximately 1 uM each 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate are used.
Following amplification, reaction are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~o ~~iooaa~ 2 ,~ ~, ~ ~ ~ ~ Pcrius9z'o~a7~
Double gap LCR EDG3,2:2 dNTPs) is performed for 30-SO cycles as in the previous examples. The oligonucleotides used are listed in Table IV, and are specific for map positions 2683-2730 within the gag region of the HIV 1 genome (HIV
isolate). Reaction conditions are similar to those described in Examples t -3, except that approximately S x 10 i > molecules each of oligonucleotides 2683. ! , 2683.2, 2683.3, 2683.4 (ID Nos. 37-40, respectively>, and approximately 1 uM each 2'-deoxyadenosine 5'-triphosphate and 2'-deoxyguanosine S'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with Ihlx diluent buffer and amplification products are detected vfa a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~Q~le 11.
Double gap LCR (DG3,3:2dNTPs) is performed for 30-SO cycles using the t S oligonucteotides listed in Table IV. These oligonucleotides are specific for map positions f 753-t 800 within the gag region of the HIV 1 genome (HIV SF2CG
isolate>. Reaction conditions are similar to those described in Examples t -3, except that approximately 5 x 10i 1 molecules each of oligonucleQtides 1753.1, 1753.2, 1753.3, 1753.4 tID Nas. 41-44, respectively), and approximately 1 ~M 2'-deoxythymidine S'-triphosphate and 2'-deoxycytidine 5'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~=xa 1~I?_.
Double gap LCR (DG3,3> is performed for 30-SO cycles as in the previous examples. The oliganuGieotides used are listed in Table IV, and are specific for map positions 3697-3744 w itf,in the poi region of the HI V t genome (Hi V t SF2CG
isolate) except that the nucleotide in position 17 of oligonucleotide 3697. i was changed from T to C to disrupt a possible intramolecular hairpin structure (position S in oligonucleotide 3697.2 changed from A to G). Reaction conditions are like those described in Examples 1-3; except that approximately 5 x 101, molecules each oligohucleotides 3697:1, 3697.2, 3697.3, 3697.4 (' ~ Nos. 45-48, respectively>, and 1 pM 2'-deoxythymidine 5'-triphosphate are uses.
Following amplification, the reactions are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
A) ~~~." :',~, ' .. .. ;v:: '.~.:.
i~~ ~3>07 PC'1"/LJS32/05~?7 Examples 13-15 relate to the detection of HI V 2 DNA. The sequences, their map position according to Franchini, G., et a1. Proc. Natl. Acad Sci. 86:2433-( 1988>, and their Sequence 10 Nos. are given in Table V, below.
- ~ TABLE V
2060. t : FL-GACAGAGGAC TTGCTGCACC 49 TCAA
2060.2: GAGGTGCAGC AAGTCCTCTG TCG-FL50 2060.3: CTCTCTTTGG AAAAGACCAG TAG-BIO51 2060.4: BIO-TACTGGTCTT TTCCAAAGAG s2 AGAA
4661.1: FL-ATCACCACAG AACAAGAAAT 53 ACAA
4661.2: GTATTTCTTG TTCTGTGGTG ATC-FL54 4661.3: CCTCCAAGCA AAAAATTCAA AAT-BIO55 4661.4: Bi0-TTTTGAATTT TTTGCTTGGA 56 GGAA
1094.1: FL-CTATGATATT AATCAAATGC 57 TTAA
~ r ' 1094.2: AAGCATTTGA TTAATATCAT AGG-FL58 1094.3: GTGTGGGCGA CCATCAAGCA GCG-BIO59 1094.4: BIO-GCTGCTTGAT GGTCGCCCAC 60 ACAA
2S Double gap LCR tDG2,2> was performed for 45 cycles using cycling parameters identical to those described in Example 1. Reactions were set up in triplicate with either 0, 102, or 103 target molecules. The target DNA was a segment of HIV 2 genamic DNA cloned into pTZ 18. Negative reactions contained 1 microgram of human placental DNA. Ppsitive reactions contained the indicated number of copies of containing plasmid DNA in a background of 1 microgram of human placental DNA.
The oligonucleotides used are listed in Table V. These oligonucleotides are spdcific for map positions 2060-2109 within the gag region of the HIV 2 genome (I-IIV21 SY
isolate) except that the nucleotide in position 14 of oligonucleotide 2060.3 was changed from C to A to disrupt a possible intramolecular hairpin structure (position 9 in oligonucleotide 2060.4 changed from G to T>.: Reactions were run in a buffer identical to that described in Example 2 except that 5 x 101 1 molecules each oligonucleotides 2060.1, 2060.2, 2060.3, 2060.4 tiD Nos.49--52, respectively>, and 1 ~M 2'-deoxythymidine 5'-triphosphate were used.
Following amplification; reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected viva sandwich immunoassay performed using the Abbott I~lx automated immunoassay system.
°
W~ 93/i77 ~ ~. ~. ~ ~ s Number of target m~j$a~
0 .8.4 102 318.4 103 789.3 S
Double gap LCR (DG2,2> is performed for 30-50 cycles as in the previous examples. The oligonucleotides used are Listed in Table V, and are specific for map positions 1094-1 143 within the ga,greglon of the HIV 2 genome (HIV21SY
isolate).
Reaction conditions are like those described in Examples 1-3, except that approximately 5 x 101 1 molecules each oligonucleotides 1094.1, 1094.2, 1094.3, 1094.4 ( ID Nos. 53-56, respectively), and 1 pM 2'-deoxythymidine 5'-triphosphate are used.
Following amplification, the reactions are diluted 1:1 with IMx diiuent buffer and 1~~ ~ amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Double gap LCR (DG2,2> is performed for 30-50 cycles as in the previous examples. The oligohucleotides used are listed in Table V, and are specific for map positions 4661-4710 within the HIV 2 genome (HIV21SY isolate). Reaction conditions are like those described in Examples 1-3, except that approximately 5 x 101 1 molecules each oligonucleotides 4661.1, 4661.2, 4661.3, 4661.4 (IQ Nos.
57-60, respectively), and 1 pM 2'-deoxycthymidine 5'-triphosphate are used.
Following amplification, the reactions are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Examples 16 - 19 relate to detection of Human Papilloma Virus (HPV), types 16 and 18. The sequences and their Sequence ID Nos are given in Table VI. Map positions for type 18 (Examples 8 and 10) are found in Seedorf, K., et al.
Virology 145:181-185 ( 1985); while map positions for type 16 are found in Zhang, Y.-X., Morrison, S.G., and H.D. Caldweli; Nuc. Acids Res. 18:1061 ( 1990>.
W~ 9?1/UU4~~7 ~ ~ ~ ~ ~ ~ P~ I'/1JB92/05~~7 Table W .
631.1: FL-TTTAGAGCCC CAAAATGAAA TTCC 61 631.2: AATTTCATTT TGGGGCTCTA AA-FL 62 631.3: TTGACCTTCT ATGTCACGAG -B10 63 631.4: BIOCTCGTGACAT AGAAGGTCAA CC 64 480.1: FL-ATAATATAAG GGGTCGGTGG ACC 65 480.2; TCCACCGACC CCTTATATTA T-FL 66 480.3; TCGATGTATG TCTTGTTGCA GA-B10 67 480.4; BIO-TCTGCAACAAGACATACATC GACC 68 488.1: FL-CAACATAGCT GGGCAC'fATA GAGG69 488.2. TCTATAGTGC CCAGCTATGT TG-FL 70 488.3; AGTGCCATTC GTGCTGCAAC C-B10 71 488.4; BIO-GGTTGCAGCA CGAATGGCAC TGG 72 6604.1 FL-TTTGTTGGGG TAATCAATTA TTTGTT73 ,: - ~ 6604.2 CAAATAATTG ATTACCCCAA CAAA-FL 74 6604.3 CTGTGGTTGATACCACACGC A-BIO '75 6604.4 B10-TGCGTGTGGT ATCAACCACA GT 76 Example 16.
Double gap LCR was performed for 35 cycles using the same cycling parameters described in Example 1. Reactions were set up in with either 0 or 102 target DNA
molecules. The target DNA was full length HPV 18 genomic DNA cloned into pGEM3.
Negative reactions contained 15 nanograms of calf thymus DNA. Positive reactions contained 102 HPV 18 target DNA molecules in l5 nanograms of calf thymus DNA.
The oligonucieotides used are listed in Table Vl. These oligonucleotides are specific for map positions 631-676 in the E6/E7 region of the I~iPV 18 genome 3.
Reactions were run in a buffer identical to that described in Example 1 except that 5 x molecules each oiigonucleotides 631.1, 631.2, 631.3, 631.4 (ID Nos.
61°64, respectively) and t pM 2'-deoxyguanosine 5'-triphosphate were used.
Following ampltficatioh, reactions were diluted t:1 with IMx diiuent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
1~ t~L ~ e~Qlecules B~, 0 6.8 1 p2 ~ 166.6 WO 93/UiD447 , PCT/LJS92/OS477 ~1~.~~2 Fxam~le 17.
Double gap LCR (DG2,2) was performed for 35 cycles using reaction conditions .
identical to those described in Example 3. Reactions were set up in with either 0 or 102 target DNA molecules. The target DNA was full length HPV i 6 genomic DNA
cloned into pSP65. Negative reactions contained 15 nanograms of calf thymus DNA.
Positive reactions contained 102 target HPV 16 DNA molecules in 15 nanograms of calf thymus DNA. The oligonucleotides used are listed in Table VI, These oligonucleotides are specific for map positions 480-526 within the E6/E7 region of the t-IPV 16 genome. Reactions were run in a buffer identical to that used in Example 1 except that 5 x 101 1 molecules each oligonucleotides 480.1, 480.2, 480.3, 480.4 (iD Nos. 65-68, respectively>, and 1 uM 2'-deoxyguanosine 5'-triphosphate were used.
Fot,lowing amplification; reactions were diluted 1:1, with IMx diluent buffer, and ,, r ~ the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
N~!f bn ~r of target JCtt~~g ~~ fi~
0 5.9 102 28.6 E,~.m~ 1 a ~ 8.
Double gap LCR tDG2,2> was performed for 35 cycles using the same cycling parameters described in Example 1. Reactions were set up in duplicate with either 0 or 106 target DNA molecules. The target DNA was full length HPV 18 genomic DNA
cloned into a plasmid: Negative reactions contained 15 nanograms of calf thymus DNA.
Positive reactions contained 106 target HPV 18 DNA molecules in 15 nanograms of calf thymus DNA. The oligonucieotides used are listed in Table VI. These oligonucleotides are specific for map positions 488-534 within the E6/E7 region of the HPV 18 genome. Reactions were run in a buffer identical to that described in Example 1 except that 5 x t011 molecules each otigonucleotides 488.1, 488.2, 488:3, 488.4 tID Nos. 69-72, respectively), and 1 uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification; reactions were diluted 1:1 with if1x diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
VVCD 9310047 ~ ~ ~ ~, ~ ~ ~ PC'~'l~JS92/OS~''~
_34_ b.r of ~~~et molecules p 7.0 106 978.2 ~xam~~ 19.
. Double gap LCR tDGl,2) was performed for 40 cycles consisting of a 65 second incubation at 88'C and an 80 second incubation at 50'C. Reactions were set up in triplicate with either 4, 104, or 106 target molecules. The target DNA was full length viral genomes cloned into various plasmids. Alt reactions contained 330 nanograms of human placental DNA. Positive reactions contained 104 or 106 molecules of NPV 6, 16, 18, 31, 33, or 61. The oligonucleotides used are listed in Table VI. These oiigonucleotides correspond to map positions 6604-6651 in the HPV
16 genome, but do not match this sequence exactly. Nucleotide changes representing ~.:- ~ consensus between all of the HPV types listed above were introduced during synthesis. .
Reactions were run in a buffer identical to that described in Example 2 except that 4 x 1012 molecules each of oligonucleotides 6604.1, 6604.2, 6604.3, 6604.4 tID
Nos. 73-76 respectively), i 8;000 units of Thermos thermaphilus DNA ligase, 4 units of Thermos DNA polymerise (MBR); 1 OuM NAD, and l.7uM 2'-deoxyadenosine S'-triphosphate were used. Reaction volume was 200 ul with no mineral oil overlay.
Following amplification, LCR reaction products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Rate tc/s/s) tai ~ ~, ~ .~lP.~ t.~. tl~l molecules Tvae 66 Tvae L~ TYQe 18 ~~.J.. .LYE-.~ ~.1 0 ~ 6.6 6.6 6.6 6.6 6.6 6.6 104 ~ 355.4 31.4 15.3 66.9 78.8 7.4 1 Ob ~ 2 t 73. 7 1 163.8 805.5 1439.7 1597.8 62.1 Examples 20 - 21 relate to detection of ~ierpes Simplex Virus tf~SV). The sequences used; their map position according to McGeoch; D.J. et al. J. Gen.
Virol.
68;19-38 t 1987>, and their Sequence ID Nos. are given in Table V.
~c~ ~~i~7 ~ ~. ~ ~ ~ ~ ) >P~ri~~9zaosa~~
Table V! I.
475 ) .1; FL-CCCCCTGTTC TGGTTCCTAA 77 CGG
475 ) .2: GTTAGGAACC AGAACAGTGG GGA-FL.78 475 ) .3: TCCCCTGCTC TAGATATCCT CT-BIO79 475 ) ,4: BIO-GAGGATATCT AGAGCAGGGG 80 AGG
0 6465. ) : F!_-TATGACAGCT TTAGCGCCGT 8 i CAG
6465.2: TGACGGCGCT AAAGCTGCAT AG-Fi"82 6465.3: GAGGATAACC TGGGGTTCCT GAT-BIO83 6465.4: BIO-TCAGGAACCC CAGGTTATCC 84 TCG
i 5 ~"~t1!~ n 1 a 2 0.
Double gap LCR (DG2,2) was performed for 60 cycles consisting of a 60 second incubation at 85°C and a 60 second incubation at 50°C. Reactions were set up in duplicate with either 0 or ) 02 target DNA molecules. The target DNA was a 4538 by ~ f , segment of the HSV 2 genome cloned into pUC ) 9. Negative reactions contained 10 20 nanograms of human placental DNA. Positive reactions contained 102 H5V 2 plasmid molecules in ) 0 nar~ograms of human placental DNA. The otigonucleotides used are listed in Table VII. These oligonucleotides are specific for map positions 475)-4798 within the US4 region of the NSV 2 genome 2. Reactions were run in a buffer identical to that described in Example ) except that oligonucleotides 475).1, 475).2, 25 4?51.3, and 475 ) .4 ( i D Nos. 77-80, respectively) at 5 x ) 0 ~ ) molecules each per reaction, and ) uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted ):) with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay perfarmed using the Abbott IMx automated immunoassay system.
r of t~C eat mold .
p 6.0 102 2096.0 Double gap LCR (DG ) ,1 ) was performed for 60 cycles using cycling parameters identical to those described in Example t. Reactions were set up in duplicate with either 0 or 102 target DNA molecules. Negative reactions contained 10 nanagrams of human placental DNA. Positive reactions contained ) 02 HSV target DNA
mo)ecules in q0 ) 0 nanograms of human placental DNA. Tlie o)igonucleotides used are listed in Table V19. These oligonucleotides are specific for map positions 6465-65)2 at the genome. Reactions were run in a buffer identical to that described in Example ) except that oligonucleotides 6465.1, 6465.2, 6465.3, and 6465.4 (ID Nos. 81-84, respectively) at 5 x 101 1 each per reaction and 1 uM 2'-deoxycytidine 5'- .
triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx dituent buffer, and LCR reaction products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number o~ target mQiecules $~g 0 6.0 102 1 156.0 Examples 22 - 24 relate to detection of Chlamydia trachomatis DNA. The sequences used and their Sequence ID Nos. are given in Table VI I. The map positions for the MOMP region (Examples 1 1 and 12) are according to 8aehr, W. et al.
Proc.
Nati. Acad. Sci. USA 85:4000-4004 ( 1988) fEMBL Genbank Data Base:
Intelligenetics, Accession *J03813]; while the map positions for the cryptic plasmid region (Example 13) are according to Hatt, C. et a1. Nuc. Acids Res.
16:4053-4067 ( 1988).
Table VIII.
36. i : FL-TTTTACTTGC AAGACATTCC TCAGG85 36.2: TGAGGAATGT CTTGCAAGTA AAAGC-FL86 36.3: ATTAATTGCT ACAGGACATC TTGTC-BIO87 36.4: BIO-CAAGATGTCC TGTAGCAATT ~
552.1: GGGAATCCTG CTGAACCAAG 89 552.2: TTGGTTCAGC AGGATTCCC 90 552.3: TTATGATCGA CGGAATTCTG TG 91 552.4: CACAGAATTC CGTCGATCAT AAGG , ~2 6693.1: FL-GATACTTCGC ATCATGTGTT CC 93 6693.2: AACACATGAT GCGAAGTATC -FL 94 6693.3: AGTTTCTTTG TCCTCCTATA ACG-BIO 95 6693.4: BIO-CGTTATAGGA GGACAAAGAA ACTCC 96 Double gap LCR (DG2,2) was performed for 35 cycles using cycling parameters identical to those described in Example 3. Reactions were set up in duplicate with either 0 or 102 target DNA molecules. The target DNA was purified Chlamydia trachomatis genomic DNA. Negative reactions contained 330 nanograms of human placental DNA. Positive reactions contained 102 Chlamydia target DNA molecules in * Trade-mark ~4 93/00447 '~ ~ ~ ~" ~ ~ ~ ~ ~ PC'T/U~92105477 330 nanograms of human placental DNA. The oligonucieotides used are listed in Table Vlll. These oliganucleotides are specific for map positions 36-89 within the major outer membrane protein (MOMP) gene. Reactions were run in a buffer identical to that described in Example 2 except that 5 x 101 1 molecules each oligonucleotides S 36.1, 36.2, 36.3, 36.4 tID Nos. 85-88. respectively) and t uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with lMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
1a ~mne of a~~mol .ec~les .B~t,~.
0 7.2 102 34.2 15 EXa l~ a 23.
Double gap LCR (DG 1,2) was performed for 40 cycles consisting of a 60 second incubation at 85°C and a 60 second ihcubation at 59'C. Reactions were set up in dqplicate with either O or 102 target molecules. Negative reactions contained nanograms of human placental DNA. Positive reactions contained i 02 Chlamydia 20 target DNA molecules in 330 hanograms of human placental DNA. The oligonucteotides used are listed ire Table Vll I. These otigonucleotides are specific for map posftions 552-595 within the MOMR gene. Reactions were run in a buffer identical to that described in Example 3 except that 8 x 101 1 molecules each of oligonucieotides 552.1 552.2 552.3 552.4 (ia Nos. 89-92, respectively), and 1 >aM oc 32P
labelled 25 ' 2'-deoxycytidihe 5'-triphosphate are used.
Following amplification, the reaction products were separated by electrophoresis on a 10% polyacrylamide gel with B.OM urea at 330 v constant for about 2 hours.
The gels were then autoradiog~aphed for about l2 hours with intensifying screens toptionaT>. In the presence of target; autoradiography revealed bands equitable with 30 the extended, unligated probe, and the longer, ligated product. The negative control gave no discernible bands.
. ExamQle 4.
Double gap LCR (D~2,~) was performed for 40 cycles consisting of a 60 second 35 incubation at 85'C and a 60 second incubation at 59'C. Reactions were set up in duplicate with either O or 102 target'molecules. Negative reactions Contained nanograms of human placental DNA. Positive reactions contained 102 Chlamydia target DNA molecules in 330 nanograms of human placental DNA. The oligonucteotides v~o ~~~oo~~ ~~ -~ 1 ; n ~ :~ ~cr~us9zios~~~
_ :. ,.'i ;~
used are listed in Table VII I. These oligonucleotides are specific for map positions 6693-6739 within the Chlamydia trachomatis cryptic plasmid. Reactions were run in a buffer identical to that described in Example 3 except that 8 x 1011 molecules each oligonucleotides 6693.1, 6693.2, 6693.3, 6693.4 (ID Nos. 93-96 S respectively>, and 1 ~M 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott iMx automated immunoassay system.
Number o~"~Lg~t mote~u_le~ 8~
0 4.8 102 1538.8 Examples 25 - 29 relate to detection of Neisseria ~onorrhoeae. The sepuences used are given in Table IX. The map positions for the laz region (example 25) are according to Gotschlich, E. C.; and M. E. Seiff. FEMS Microbicl. Letts. 43:
( 1987). The map positions for the P.it/opa region (examples 26 & 27) are according to Stern, A., M. Brown, P. Nickel, and T. F. Meyer. Cell 47: 61-71 ( 1986). The map positians for the pilin region.(examples 28 & 29) are according to Meyer; T. F., E. Biliyard, R. Haas, S. Storzbach, and M. So. Proc. Natl. Acad.
Sci. USA
81: 61 10-61 14 ( 1984).
~
27.1: FL-AATATTTGCG CGGCGGGGAC GG 97 27:2: GTCCCCGCLG ~GCAAATATT -FL 98 laz 27:3: TGCCCTAATA TTAAAGAAAT AGGTT-BIO 99 27:4: BIO-AACCTATTTC TTTAATATTA GGGCAG 100 66.1: FL-GCCATATTGT GTTGAAACAC CGCCC 101 66.2: 'CGG'fGTTTCA ACACAATATG GC-FL 102 opa 66.3: AACCCGATAT AATCCGCCCT T-BIO 103 66.4: ' B10-AAGGGCGGAT TATATCGGGT TCC 104 1 14.1: FL-CAACATCAGTG AAAATCTTTT TTTAACC 105 1 14.2: TTAaAAAAAG ATTTTCACTG ATGTTG-FL 106 opa 1 14.3: TCAAACCGAA TAAGGAGCCG A-BI0 107 1 14.4: 810-TTCGGCTCCT TATTCGGTTT GACC 108 i1~~3 93/7 ~ ~ P(.°1'/gJS92/(DS477 822. i : FL-TGATGCCAGC TGAGGCAAAT TAGG 109 822.2: TAATTTGCCT CAGCTGGCAT CA-FL 1 10 pi 1 i n a 822.3: TTAAATTTCA AATAAATCAA GCGGTA-BIO1 1 1 S 822.4: BI0-TACCGCTTGA TTTATTTGAA ATTTAAGG1 12 933.1: FL-CGGGCGGGGT CGTCCGTTCC 1 13 933.2: AACGGACGAC CCCGCCCG-FL 1 14 pilin 933.3: TGGAAATAAT ATATCGATI'-BIO 1 15 933.4: BIO-AATCGATATATTATTTCCAC C 1 16 a The sequences shown are a consensus of all published N, gonorrhoeae pitin genes and when taken in total do not exactly match any one particular sequence.
~x~m~rle 25.
Double gap LCR (DG2,1 ) was performed for 35 cycles consisting of a 30 '~ second incubation at 85'C and a 60 second incubation at 5~d°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. All NeisseriaDNA's were tested in the presence of 324 ng human placenta DNA. The oligonucleotides used (see Table IX, above) are specific for map positions 27-74 within the Neisseria gonorrhoeae lay gene. Reactions were run in a buffer identical to that described in Example 3 except that 5 x 101 1 molecules each of oligonucleotides 27.1, 27.2, 27.3, 27.4 (ID Nos. 97-100, respectively), and 1 ~M 2'-deoxycytidine S'-tr~phosphate w ere used.
Following amptific~tion, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
pig o y~e (Amount) Neisseria gonorrhoeae (200 cells) 576 Neisseria meningitidis (2 x 106 cells) 6 3E, Neisseria lactamica (2 x 106 cells) 5 Human placenta (320 ng) ~'~,~~ 1R
Double gap LCR (D~3,2~ f~as performed for 27 cycles consisting of a 30 40 second incubation at 85'C and a 60 second incubation at 57°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. All Neisseria DNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX; above) are specific for map positions 66-1 13 within the Neisseria gonorrhoeae opa gene. Reac ions were run in a buffer identical to that described in 1~0 93/4044? ~ ~ ~ ~. ~ ~ ~ ' , PG'r'1US92/05~~7 Example 3 except that S x 101 1 molecules each of oligonucleotides 66. t , 66.2, 66.3, 66.4 (ID Nos. 101-104, respectively), and 1~M 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactiorys were diluted t:i with IMx diluent buffer, S and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
_Dj~A ~ourr.e ~~ o m ) Neisseria ganorrhaeae 1200 cells) t ~ 10 Neisseria meningitides (2 x 106 cells) 18 Neisseria lactamica (2 x 106 cells) 8 Human placenta (320 ng> 6 E-x~OBle 2'7.
Double gap LCR (DG2,2> vvas performed for 35 cycles consisting of a 30 second incubation at 85°C and a 60 second incubation at 54°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. Ati NeisssriaDNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX, above) are specific for map positions 114-16S within the Neisseria gonorrhaeae opa gene. Reactions were run in a buffer identical to that described in Example 3 except that S x 101 1 molecules each of oligonucleotides 1 14.1, 1 14.2, t t 4.3, t 14.4 ( I D Nos. t 05-108, respectively), and 1 uM 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification pr~ducts were detected via a sandwich immunoassay performed using the Abbott iMx automated immunoassay system.
ANA o ~,~~5,~~"ount) B, Neisseria gdnarrhoeae (200 cells) 306 Neisseria meningitides (2 x 106 cells) 10 Neisseria lactamica (2 x 106 cells) 10 Human placenta (320 ng) ~.Im~le-2_~- ' Double gap LCR (DG2,2> was performed for 35 cycles consisting of a 30 second incubation at 85°C-and a 6Q second incubation at 50°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. A11 Neisseria DNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX; above) are specific for map positions 822-873 within the Neisseria gonarrhoeae piiin gene. Reactions were run in a buffer identical to that described in w~ q~iooa~~ ~ ~ ~ ~ ~ ~ ~ Pc-rius9~oosa~'~
Example 3 except that 5 x i 01 1 molecules each of oligonucleatides 822.1, 822.2, 822.3, 822.4 (ID Nos: 109-1 12, respectively), and 1~M 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx dtluent buffer, S and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~N ~~,~ . .fount ) 8 Neisseria gonorrhoeae (200 cells) 1291 Neisseria meningitides (2 x 106 cel)s) 27 Neisseria lactamica (2 x t 06 cells) 22 Human placenta (320 ng) 1 1 1 S ~xa y a 29.
~; ~ . Double gap LCR (DG2,2> is performed for 30 - 50 cycles as in earlier examples. The oligonucleotides used (~~~e Table iX, above) are specific for map positions 933-973 within the Neisseria gonorrhoeae pitin gene. Reactions are run in a buffer identical to that described in Example 3 except that approximately 5 x 101 1 molecules each of oligonucleotides 933.1, 933.2, 933.3, 933.4 (ID Nos. 1 1 16, respectively), and 1 uP~4 2'-deoxyguanosine 5'-triphosphate are used.
Following amplification, reactions are diluted 1:1 with IMx diluent buffer, and the LCR amplification products are detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Examples 30 ahd 31 relate to the detection of Sorrel is burgdarferi, the causative agent of "Lyme'° disease. The sequences used, their map position according to Rosa; P,A., and T:G. Schwan. J: Infect. Dis. 160:1018-1029 (1989), and their Sequence iD Nos. are: given in Table X, below. .
Table X:
~1~P.PQ S SEA EU NCE- SEG? I
D No.
S. t : FL-AAAACGAAGA TACTAAATCT GTAA1 17 5.2: ACAGATTTAG TATCTTCGTT TT-FL 1 18 5.3: CCAGAAACAC CTTTTGAATT AA-SIC!1 19 5.4: B60-TTAATTCAAA AGGTGTTTCT 120 GCAA
181.1: FL-CATCTTTTGG AGCTAAATAT AAG i 2 l 181.2: TTATATTTAG CTCCAAAAGA.TGC~FL 122 181.3: TTGGATTAAC AAAAATAAAC GAT-BIO123 181.4: BIO-TCGTTTATTT TTGTTAATCC 124 AAG
.~.,: , ;., '.;, . ...:.; - :. ;:v ~:_ . ~ . :. ::"
PCT/LJ~92/05~'~7 dVfJ 93/0047 Double gap LCR (DG2,2> was performed for 45 cycles consisting of a 25 second incubation at 8S'C and a 70 second incubation at 50'C. Reactions were set up in S duplicate with either 0'or 102 target molecules. The target DNA was a random Borrelia burgdorferi genomic sequence cloned into pUC 18. Negative reactions contained 10 nanbgrams of calf thymus DNA. Positive reactions contained 102 Borrelia target DNA molecules in 10 nanograms of calf thymus DNA. The oligonucleotides used are listed in Table X, and are specific for map positions 5-53 of the cloned Borrelia genomic DNA. Reactions were run in a buffer identical to that described in Example 1 except that 3 x 10~? molecules each otigonucleotides 5.1 and 5.3 tID Nos. 1 i7 and 1 19, respectively), 2 x 102 molecules each otigonucleotides 5.2 and 5.4 (!D Nas. 1 18 and120, respectively>, 1032 units of Thermos thermophilus DNA lipase, and 1 uM 2'deoxythymidine 5'-triphosphate were used.
. Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number of target molecules 0 6.0 i 02 152.0 Double gap LCR (DG 1,1 > was performed for 45 cycles consisting of a 65 second incubation at 85°C and a 60second incubation at 50°C. Reactions were set up in duplicate with either 0 or 102 target molecules. Negative reactions contained nanograms of salmon sperm DNA. Positive reactions contained 102 8orrelis target ~NA moteeuies in t 0 nanograms of salmon sperm DNA. The oligonucleotides used are listed in Table X; and are specific for map positions i 81-228 of the cloned Borrelia genamic DNA. Reactions were run in a buffer identical to that described in Example 1 except that 1 x 10~~ molecules each oligonucleotides 18i.1, 181.2, 181.3, 181.4 E!D Nos. i21-124, respectively), 500 units of Thermos thermophilus DNA lipase, 1.6 units of Thermos DNA polymerase (MBR), and t p.M 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with !Mx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~~ g~~o~a~ ~ ~. ~. ~. ~ ~ 2 Pcrius~~i~sa~' Number of ar~i' mot~c~.~l~~ Vie.
p S.0 319.0 The above examples serve to illustrate the 'invention, but are not intended to limit it in any way, the felt scope of the tnvention being dettned by the appended claims.
i,r' r V4~~ ~3/0~447 I'CT/~JS92/0~~77 A.
AZVSF2CG Subxequence Sites Tbusxday, June a?, 1991 8:40 AH
Sequence Range: 991 to 2299 Strt #Res.
Bos. Subseq Ioc. Hatched Subseq Found ,Search with DG 5 sites 2,2 (A): $oundm 811 ~G 2v2 (A) (part #1) 811 B/6 ATTAAG
+
816 DG 2,2 (A) .. , ~
(part #1) 816 6/6 CETAAT
~
850 DG; 2.2 (A) (part #1) 850 6/6 ~rxAAG
+
e55 ~c z,z (A) (part #1) 855 6/6 CTTAAC
~
1964 DC 2,2 (A) (part #1) 1964 6l6 GTTAAG
+
1969 35C 2,2 (A) (Past ~Il? 1969 6l6 CTTAAC
.
Search wl.th DG 14 2.2 (C)a sites found..
854 DO; 2,2 (C) (past 1~1) 854 6/6 ACGCCA
+
859 ~ 2' Ip~. #1) 059 6/6 TCGCCT
-934 DG 2,2 (C) -(part #1) 934 6/6 T:.GCCT
+
939 ~c z,z (c) (part #1) 939- 6/6 ACGCCA
-123a ~ z,2 (c) (part #1) 1230 s/s A~cccA
+
1235 d7G 2,2 (C) (pazt #1) 1235 6/6 TCGCCT
-laal ~c z,a (c) (part #1) 1471 6/6 31GGCCA
+
1496 DG 2.2 ICD _ ~ v .
(part #1) '1476 6/6 TJGCCT
-1864 DG 2,2 (C) (part #1) 1864 6/6 CGGCCA
+
1$69 DG 2v2 (C) (part #1) 1869 6/6 TCGCCC
~-2108 TsG 2.2 (C) (part #1) 2108 6/6 3'GGCCT
~+
2113 DG 2,2 (C) (part #1) 2113 6/6 AG~GCCA
-2126 DG 2,2 (C) (paict #I) 2126 6/6 AGGCCA
+
2131 DG 2,2 (C) (part #1) 2231 6/6 ~'GGCCT
~-Sea=ch vith DG 2 sites 2,2 (G), founcL
1689 DG 2,2 (G) (part #1) 1689 6/6 ACCCCT
+
1694 DG 2,2 (G) (part #1) 1694 , s/6 ACCGGT
-Search with DC 4 sites 2,2 (T): found_ 824 L7C 2,'2 (T) (past #1) 824 6l6 G~1TTA
+
829 DC 2.2 (T) (part #i) sag s/s TAaTTc -1975 DG 2,2 (T) (part #1) 1975 6/6 c..l:,TTG
+
1980 I7G 2,2 (T) (part #1) 1980 6/b CAATTG
-APPENDIX A
Targets are selected and probes are designed so that only one, or a maximum of two, of the four deoxyribonucleotide triphosphates is needed to fill both the gaps.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a method for detecting the presence of a target nucleic acid sequence in a sample, said method employing the ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target sequence, said method comprising:
a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula:
S'-(N)hXEp.FqZ(N)k-3' wherein E represents any base, F represents any base except E, p and q are independently integers from 1 to about 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k> each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected;
b) providing a plurality of each of four probes having the formulas:
A 5'-(N)h~ X Ep-3' A': 3'-(N')htX'-5' B: 5'-Z (N)k~-3' B': ~ 3'-F'qZ'(N')k~-5' wherein the symbols have the same meanings given above, except that h' and k' need only approximate h and k, respectively, varying by no more than 25~, and wherein at least one of the probes is labeled with a moiety capable of detection; and also providing deoxyribonucleotide triphosphates of E' and F, a polymerase reagent and a ligase reagent;
c) performing the following cycle at least once:
i> mixing said probes with said sample under hybridizing Conditions to allow probes to hybridize to the target sequence and'its complement if present, or to reorganized probes created therefrom;
i i ) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F
', ~cr~u~9~~o~ ._., Wo 93~dDO447 deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerise reagent try adding E' deoxyribonucleotide triphosphates to Its 3' end iii»igating extended probe A to probe B, and extended probe B' to probe A', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
d) separating reorganized probe molecules from unreorganized labeled probes; and e) detecting the presence of said label in the reorganized or fraction as a measure of the presence of the target sequence.
In especially preferred embodiments, F is E' so that both gaps can be filled by the deoxyribonucleotide triphosphate of only E'. Preferably p and q are small, ie., between 1 and 3, inclusive. Integers p and q may be equal ar may differ try one or more. Preferably, the label comprises one or more haptens covalently bound to at 1 S least one of the probes.
In another aspect, the invention relates to diagnostic kits for the detection of a target nucleic acid sequence in a sample, said nucleic acid having a target sequence comprising 5'-(N)hXEp.FqZ(N)k-3' wherein E and F independently represent any base, p and q are independently integers from 1 to about 10, X represents any erase except E or F', Z represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p*h> and (q*k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic far the target sought to be detected, said kit comprising in combination:
(a) four probes, having the formulas:
A: 5'-f N)ht X Ep-3, A': 3'-( N' )I,~X'-5' B: 5~-Z (N>~~-3, B'; 3'-F'qZ'tN')ky-5' wherein the symbols have the same meanings given above, except that h' and k' need only approximate h and k, respectively, varying by no more than 25~, and wherein at least one of the probes is labeled with a moiety capable of detection;
(b) a polymerise reagent capable of extending probe A with said polymerise ~5 reagent in a target dependent mahner by adding F deoxyribonucleotide triphosphates to its 3' end; and extending probe B' with said polymerise reagent in a target dependent manner by adding E' deoxyribonucleotide triphosphates to its 3' end, thereby to render the primary probes iigatable to one another when hybridized to target and, ~.. :.. , : . ,. .; <;.. . .~,;;.. . :;: . ,, , ; , ,; :,: .., .;, .,.,.
vV~ 93!00447 ~ ~ ~ ~ ~ ~ ~ P~('/US921054'77 optionally, to render the secondary probes ligatable to one another when hybridized to target;
(c> deoxyribonucteotide triphosphates of E' and F; and td) a ligase reagent for ligating the extended probe A to probe B, and extended probe S' to probe A', thereby to form reorganized probe molecules.
Especially preferred kits, also include means for separating reorganized probe molecules from unreorganized labeied probes and/or means for detecting the detectable label.
In yet another aspect, the invention relates to compositions of matter each comprising a mixture of four probes, the four probes being selected from specific . sets of sequences given in the examples. These compositions of matter have utility in detecting the presence of certain pathogens according to the methods described above.
It will, of course, be realized that sequences being slightiy shorter or longer than the ~, ~ ~ exemplified and claimed sequences are deemed to fall within the scope of the 1 S invention, provided they are capable of hybridizing with the same locations on the targets as the claimed sequences.
EiRIEF DESCRIPTION OF THE DRAWINGS
Figure t is a graphic representation of the process of ligase chain reaction as ~0 it is known in the prior art.
Figure 2 is a graphic representation of a generalized, double gap variation of the invention.
Figure 3 is a graphic representation of a somewhat more specific double gap variation of the invention.
W~J 93J~0447 '~ ~ i .,~' .,. .- ~' PCf/US'~2/O5~'"7 DETAILED DESCRIRTlON
For purposes of this invention, the target sequence is described to be single stranded. However, this should be understood to include the ease where the target is actually double stranded but is simply separated from its complement prior to hybridization with the probes. In the case of double stranded target, the third and fourth (secondary) probes, A' and B', respectively, will participate in the initial step by hybridizing to the target complement. In the case of single stranded target, they will not participate in the initial hybridization step, but wail participate in subsequent hybridization steps, combining with the primary fused sequence produced by ligating the first and second probes. Target sequences may comprise deoxyribonucleic acid (DNA> or ribonucleic acid (RNA).
Target sequences may be the nucleic acid of virtually any entity which contains nucleic acid. For example, but not limitation, the bacteria and viruses ~, r ~ illustrated in the examples have nucleic acid target sequences. Target sequences may, but need not, represent pathogenic organisms or viruses. Target sequences may also represent nucleic acid which is being examined for the presence of a specific allele, or for a specific mutation of deletion, such as is often the case in genetic testing.
Finally; target sequences may be simply any nucleic acid sequence of general interest, such as may be the case in forensic analysis of DNA.
Throughout this application, the "prime" ('> designation is used to indicate a complementary base,or sequence, A probe is "complementary" to another probe if it hybridizes to the other probe and has substantially complementary base pairs in the hybridized region, Thus, probe A can be complementary to A' even though it may have ends not coterminal with A': The same is true of B and B'. Similarly, the short sequences Xn and Ym have complementary sequences designated a~ X'n and Y'm, respectively. Finally, the complement of a single base, e.g. Q, is designated as Q'. As used herein with respect to sequences;''complementary" encompasses sequences that have mismatched base pairs in the hybridizable region, provided they can be made to hybridize under assay conditiohs.
It is also to be understood that the term "the 4 bases" shall refer to Guanine (G), Cytosine tC), Adenine (A) and Thymine (T) when the context is that of DNA; beat shall refer to Guanine (G), Cytosine (C), Adenine (A) and Uracil (U) in the context of RNA. The term also includes analogs and derivatives of the bases named above.
Although the degenerate base lnosine (I) maybe employed with this invention, it is not preferred to use l within modified portions of the probes according to the invention.
It is an impørtant feature of the present invention that instead of using two pairs of probes capable of forming blunt-ended duplexes, at least one probe of one of the probe pairs initially includes a "modified" end which renders the resultant duplex "nonblunt" and/or not a suitable substrate for the ligase catalyzed fusion of the two probe duplexes. A "modified end" is defined with respect to the point of ligation rather than with respect to its complementary probe. A "modified end" has omitted bases to create a "gap" between one probe terminus and the next probe terminus (See, e.g., probes A' and B, of Figs. 2 and 3:) By convention in this application, a modified end is referred to herein as a "recess", the recess being the gap between two primary or secondary probes after hybridizing to the target. The presence of these modified ends reduces the falsely positive signal created by blunt-end ligation of complementary probe duplexes to one another in the absence of target.
"Correction" of the modification is subsequently carried out to render the probes ligatable. As used herein "correction" refers to the process of rendering, in a target dependent manner, the two primary probes or the two secondary probes ligatable to their partners. Thus, only those probes hybridized to target, target complement or polynucleotide sequences generated therefrom are "corrected."
"Correction" can be accomplished by several procedures, depending on the type of modified end used.
As used herein, "point of ligation" or "intended point of ligation" refers to a specific location between two probe partners that are to be ligated in a template-dependent manner. It is the site at which the "corrected" probe lies adjacent its partner in 3' hydroxyl-S' phosphate relationship. For each set of four LCR
probes there are two "points of ligation", a point for the primary probe partners and a point for the secondary probe partners. In conventional LCR the two points of ligation are opposite one another, thus forming blunt ended duplexes when the probe pairs hybridize to one another. In the present invention, the points of ligation are not opposite one another; but are displaced from one another in the "recess"
embodiments by one or more bases by virtue of the gaps. The exact point(s> of l;igation varies depending on the embodiment and, thus, this term is further defined in the context of each embodiment.
Each of the probes may comprise deoxyribonucleic acid (DNA) or ribonucleic a~~'~i (RNA). It is a routine matter to synthesize the desired probes using c ventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, CA); DuPont, (Wilmington, DE); or Milligen, (Bedford, MA>. Phosphorylation of the S' ends of the appropriate probes (eg. A' and B), while necessary for ligation by ligase, may be accomplished by a kinase or by commercial synthesis reagents, as is known in the art.
* Trade-mark ~rV~ 93/40~i447 ; r . ~ . ' , PC~'/US~2/O5a''~
'~'.~~~~(~~
Throughout this application, the bases X, Y and Q, and their complements are described as being selected from certain subsets (P! or M> of the 4 bases. In reality, the sequences are not "selected" at all, but are dictated by the sequence of the target strand. The term "selected" in this context is taken to mean that a target sequence having the desired characteristics is located and probes are constructed around an appropriate segments) of the target sequence, In general, the methods of the invention comprise repeated steps of (a) hybridizing the modified probes to the target (and, 1f double stranded so.that target complement is present, to the target complement>; (b) correcting the modification in i 0 a target dependent manner to render the probes ligatable; (c) ligating the corrected probe to its partner to form a fused or ligated product; and (d> dissociating the fused product from the target and repeating the hybridization, correction and ligation steps to amplify the desired target sequence. Steps (a), (c) and (d) are essentially the same for all of the embodiments and can be discussed together. They are generally the same steps that one would employ in conventional LCR. Step (b) varies depending on the type of modification employed and each different type is discussed separately.
Hybridization of probes to target (and optionally to target complement) is adequately explained in the prior art; e.g EP-320 308. Probe length, probe concentration and stringehcy of conditions all affect the degree and rate at which hybridization will occur. Preferably, the probes are sufficiently long to provide the desired specificity; i.e, to avoid being hybridizable to nontarget sequences in the sample. Typically, probes on the order of 15 to t 00 bases serve this purpose.
Presently preferred are probes having a length of from about 1 S to about 40 bases.
The probes are added in approximately equimolar concentration since they are expected to react stoichiometrically. Each probe is present in a concentration ranging from about S nanomolar (nM) to about 90 nM; preferably from about 10 nM to about nM. For a standard reaction volume of 50~L, this is equivalent to adding from about 3 x 10 t t to about 1 x 10 ~ 2 molecules of each probe; and around S
x,10 ~ ~
molecules per 50 uL has been a good starting point. The optimum quantity of probe 30 used for each reaction also varies depending on the number of cycles which must be performed and, of course, the reaction volume. Probe concentrations can readily be determined by one of ordinary skill i~ this art to provide optimum signal for a given number of cycles.
The stringency of conditions is generally known to those in the art to be 35 dependent on temperature; solvent and other parameters. Perhaps the most easily controlled of these parameters is temperature and thus it is generally the stringency parameter varied in the performance of LCR. Since the stringency conditions required for practicing this invention are not unlike those of ordinary LCR, further '~~'O ~3/00~7 ~ ~ ~ ~ P(_'T/iJS92/05~77 _g_ detail is deemed unnecessary, the routine practitioner being guided by the examples which follow.
The next step in the general method follows the specific correction step and comprises the ligation of one probe to its adjacent partner. Thus, each corrected primary probe is ligated to its associated primary probe and each corrected secondary probe is ligated to its associated secondary probe. An "adjacent" probe is either one of two probes hybridizable with the target in a contiguous orientation, one of which lies with its phosphorylated 5' end in abutment with the 3' hydroxyl end of the partner .
probe. "Adjacent" probes are created upon correction of the modified ends) in a target dependent manner. Since enzymatic tigation is the preferred method of covalently attaching two adjacent probes, the term "ligation" wail be used throughout the application. However, "ligation" is a general term and is to be understood to include any method of covalently attaching two probes. ~ne alternative to enzymatic ligation is photo-ligation as described in EP-A-324 616.
The conditions and reagents which make possible the preferred enzymatic ligation step are generally known to those of ordinary skill in the art and are disclosed in the references mentioned in background. Ligating reagents useful in the present invention include T4 ligase; and prokaryotic ligases such as E coli iigase, and Thermus thermophi?us ligase (e.g., ATCC 27634) as taught in ~P-320 308. This latter ligase is presently preferred for its ability to maintain activity during the thermal cycling of LCR: Absent a thermal ~y stable ligase, the ligase must be added again each time the cycle is repeated. Also useful are eukaryotic ligases, including DNA lipase of Drosophilia; reported by Robin, et al., ,J. Bfol. Chem.
261:10637-10647 ( 1986 >. .
Once ligated, the fused; reorganized probe is dissociated (e.g. melted) from the targe and, as with conventional LCR; the process is repeated for several cycles. The number of repeat cycr ~s may vary from fo about 100, although from about 15 to about 70 are preferred presently. , (t is desirable to design probes so that when hybridized to their complementary (secondary) probes, tho ends away from the point of intended ligation are not able themselves to participate in other unwanted ligation reactions.
Thus, li~atable sticky or blunt ends should be avoided. If such ends must be used, then S' terminal phosphates should be avoided, eliminated w blocked. This can be accomplished either through synthesizing oligonucleotide probes (which normally carry no 5' terminal phosphate groups), or through the use of phosphatase enzymes to remove terminal phosphates (e.g, from oligonucleotides generated through restriction digests of DNA). Alternatively, ligation of the "wrong" outside ends of the probes can be prevented by blocking the end of at least one of the probes with a "hook"
~ ~ ~. ~ 9 ~ 2 ~~~U~~z~~s~~~
-lo-or marker moiety as will be described in detail below. In the absence of one of the above techniques, the outside ends of the probes can be staggered so that if they are joined, they will not serve as template for exponential amplification.
Following amplification, the amplified sequences can be detected by a number of conventional ways known in the art. Typically, detection is performed after separation, by detemining the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction. Separation may be accomplished by electrophoresis, by chromatography or by the preferred method described below.
!n a particularly preferred configuration, haptens, or "hooks"', are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of ail four probes. A °'hook" is any moiety having a ', p . specific ligand-receptor affinity. Typically, the hooks) at one end of the fused product (e.g. the 5' end of A and the 3' end of A') comprises an antigen or hapten capable of being immobilized by a specific binding reagent (such as antibody or avidin> coated onto a solid phase. The hooks) at the other end te.g. th@ 3' end of B and the S' end of B') contains a different antigen or hapten capable of being recognized by a label or a label system such as an antibody-enzyme conjugate. Exemplary hooks include biotin, fluorescein and digoxin among many others known in the art. A
substrate is then added which is converted by the enzyme to a detectable product.
EP-A-330 221 to Enzo describes oligonucleotides having a biotin molecule attached at one end.
E't'!p~MODIF~ED BY ~ -ESSE
In this embodiment; modified ends are created by eliminating from one or yore of the probes'a short sequence of bases, thereby leaving a recess or gap between the 5' end of one probe and the 3' end of the other probe when they are both hybridized to the target for target complement, or polynucleotide generafied therefrom).
In order for LCR to amplify the target, the gaps between the probes must be filled in f i.e., the modification must be "corrected"). In a first version, this can be done using a polymerase or a reverse transcriptase and an excess of deoxyribonucleotide triphosphates which are complementary to the target strand apposite the gap.
However, prior to,discussing this embodiment in detail, a brief digression on set terminology may be helpful: A set te.g., S> consists of alt the elements contained within the set S: The set "not S" then consists of all the remaining elements of the "Universe" which are not found in S. The "tJhiverse" for purposes of this application consists of the four bases G, C, A and T, ar G, C; A and U as described above.
The . Y.:. .. 4 ~'..1: n . , ..,...... . ,...,-..;,.. . .. ..,...,... . ::?~' .....z'::~. . .,~..e::
......,:;::.. .....~....., ., .:~':...:~.....,.oY. .. tu. ;:.:'. . 'a::~'i .
....r.'.::., n. ..~:,' ..,. ; .::: ,. ,. ~,~:..
intersection of set S and another set (e.g., R) consists only of those elements which are found in both S and R. Thus, as used in this application, the set "not N
and not M"
consists of those bases which are present in neither the gap Xn nor the gap Ym.
According to this invention, the set "not N and not M" must not be an empty set; i.e at least one base must remain in this set to code for the "stopbase".
Gap Filling by Extension:
In accordance with this first version, the invention involves repeated steps of (a> hybridizing the probes to the target (and, if double stranded so that target complement is present, to the target complement); (b> extending at least one probe to fill in at least one gap, designated Xn; (c) llgating the extended probe to the adjacent probe to form a fused or ligated product; and (d> dissociating the fused product from the target and repeating the hybridization, extension and ligation steps to amplify the desired target sequence.
In this version, which includes both single gap ("SG"> and double gap ("DG") configurations, the "gaps" Xn and Ym which impart the "modified ends" are "corrected" by extending one or both of the modified probes using a polymerase or a reverse transcriptase. Generally, extension of a probe hybridized to a DNA
target is accomplished by a DNA polymerase or a Klenow fragment as is known in the art.
In the case of an RNA target, extension is accomplished t a reverse transcriptase.
Exemplary reverse transcriptases include those from avian myeloblastosis virus (AMV) and Moioney murine leukemia virus (M-MuLV) generally available to those skilled in the art. Certain DNA polymerases will also recognize RNA as template under certain conditions. It is, of course, preferable to utilize extension reagents which are thermally stable and can withstand the cycling of high temperatures required for LCR. If the extension reagent is not thermally stable, it typically must be re-added at each cycle of 1.CR. Such thermostable polymerases~presently include AmpliTaqTM, (available from Cetus-Perkin Eimer), Thermos polymerase (available from Molecular Biology Resources,lnc. Milwaukee. WI, "MBR") and recombinant or purified native polymerases from Thermos aQuaticus, Thermos thermophilus or other species known to be thermostable.
Correction by extension in this manner requires the presence in the reaction mixture of deoxyribonucleotide triphosphates (dNTP's) complementary to the bases of the target in the gap region(s). More specifically, for a gap having the sequence Xn, the dNTP's that must be supplied are designated dX'TP wherein X' stands for the the complements of each base in the gap X~. The dNTP's are commercially available from a number of sources, including Pharmacia (Piscataway, ttJ) and Bethesda Research Laboratories (Gaithersburg, MD>.
* Trade-mark ~r~ ~3ioo~7 . . . ~crms~zios~~7 Extension must be terminated precisely at the point of ligation so that the extended probe abuts'the adjacent probe and can be ligated to it. "Stopbases"
are employed for this purpose, (See figure 2). A "stopbase", designated D', is defined in terms of its complement, Q and is accomplished by omitting from the reaction S mixture, dNTP's that are complementary to Q; i.e. by omitting dQ'TP from the reaction mixture. Thus it is seen how the bases for the gap sequence(s> must be selected from a set, N, consisting of only three of the four bases, so that the complementary three of the four dNTP's are added to the reaction mixture. When the fourth dNTP, dQ'TP, is absent from the reaction mixture extension will terminate at the desired point of ligation. It follows that Q' is the first base in the adjacent probe, and the base on the target which codes for the stopbase is the first base adjacent the gap (on the S' end of the X~ gap in Figure 2).
White the concept is easiest to grasp in the SG configuration, it should be understood that the SG variation is merely a special case of the double gap (DG) 1 S variation discussed below (in SG, m=0). However, only the DG configuration is relevant to the presently claimed invention. As shown in Fig 2, a first probe, A, hybridizes to a first segment of the target strand, T. A second probe, B, hybridizes to a ~ecohd segment pf: the target strand, leaving a gap of one or more bases between the two probes This gap is designated Xn on the target. Following extension and ligation, a third ps~obe, A', is hybridizable to a first portion of reorganized probe, A:B; and a fourth probe; B', is hyoridizabie to a second portion of reorganized probe, A:B. In the DG configuration, the: third probe, A', and the fourth probe, B'; hybridize such that a gyp of one or more bases lies between the probes. This gap is designated Y,~
on the reorganized probe (and on target comptemeht). As is shown in Fig. 2, the target 2S strand, T, may be double stranded, having a complement, T'. In this case, the third and fourth probes may participate in the initial hybridization by hybridizing to first and second segments'of the-target Complement.
Extension by polymerase or transcriptase proceeds in a 5' to 3' direction.
Consequently, the 3' ends of both A and 8' will be extendable by polymerase in the absence of anything to prevent extension. Extension is terminated when the next base called for by the template is absent from the reaction mixture. Thus, probe A
is extended through gap Xn until stopbase complement (Q> is encountered along the target trahd. Similarly, probe B' is extended through gap Ym until stopbase complement (Q> is encountered (either on the target complement or on the A
half of ~5 reorganized A:B>. Neither probe A' nor B will serve as a template for extension of A
Qr B'; so probes A and B' are extended only if hybridized to the target (or to reorganized polynucleotide products from previous cycles).
t. ., ° ' . ;.,. . - . ,.. ,; ;. . :. , . ~ ;
WO 93/00447 ~ ~ ~ ~ ~ PC'~'/US92/05477 As alluded to above, it is important to terminate the extension of 'A and B' at the end of the respective gaps (i.e., at the point of ligation) so that the extended probe can be iigated to the 5' end of the adjacent probes, B and A'. Therefore, the reaction mixture omits the deoxyribonucleotide triphosphate complementary to the base (a>
S immediately adjacent the S' end of gaps Xn, and Ym. Of course, it will be understood that it is not required that the same base stop extension in both directions.
A different base can be used provided it is not needed to fill either of the gaps. it should now be apparent that the actual points of ligation in this embodiment are always at the S' ends of probes A' and B. It is not by mere coincidence that these are also the locations of the stopbases Q'.
Accordingly, the gaps Xn and Ym can be any number of bases long, i.e., n can be any integer greater than or equal to 1, and m is any integer greater than 0.
It is to realized, however, that the choice of which gap is Xn and which is Ym is arbitrary in the first place; but n and m cannot both be zero. The gaps need not be the same length, ~,r' i.e., m need not equal n. When, m equals zero, the double gap variation degenerates into the specialized case of the single gap, which is not used in the embodiment being claimed herein. Tho only restriction on the bases X is that they be selected from a Set N which consists of from 1 to any 3 of the four bases. Similarly, the bases Y
are drawn from set M. Since at least one stopbase Q' must be maintained, the combined sets N and M which represent the possible bases for X and Y, respectively, must include no more than three of the four bases. Accordingly, Y can be from zero to any three of the four bases provided that at least one base remains in the set "not N and not M". If set N cor(stitutes less than three of the four bases, then Y can be a base that is not within N so long as there is at least one base remaining, the complement of which can serve as the stopbase Q' for termination of probe extension. A single stopbase can serve to terminate extension ih both the Xn and Ym gaps.
A second limitation on sequence Ym occurs if m equals n. If the gaps are the same length, the sequence Ym should not be complementary to the sequence Xn or the 3' ends of probes A and,B' would constitute "sticky ends". "Sticky ends" would permit a target independent double stranded complex to f orm wherein probe A
hybridizes to probe B' such that'ligations and amplification would proceed. Rather, when m equals n it is preferred that Ym not be complementary to X~. In other words, the ends of probes A and B' shauld at least be "slippery ends" which may be the same length, but are not complementary.
in a preferred aspect of the invention, the fourth probe B' includes a 3' terminal sequence of X~, identical in length to the Xn sequence gap in the target. This arrangement is not essential to the invention, however, as the gap need only be formed between the probes. Thus, the 3' terminus of the fourth probe B' may stop short of W~U 93I004~t7 ~ ~ ~ ~ ~ ~ ~ PCT/UB92/05~"'7 the 3' end of sequence Xn, provided there is no 3' recessed end with respect to the second probe B. Since extension occurs in a S' to 3'' direction and dX'TPs must be present anyway (to extend through Xn), probe B' would be extended through the gap, (bath Ym and any remainder of Xn > just as the first probe A is extended through the Xn gap.
The general method of the invention employing the double gap embodiment is now described briefly. First, probes A, and B are allowed to hybridize to target, if present. If the target is double stranded, it is first denatured and probes A' and B' can also hybridize to the target complement. In the presence of a suitable polymerase, probes A and B' are extended by the addition of dX'TPs and dY'TPs, respectively, to their 3' ends using the target strand as a template. ~iowever, when the polymerase encounters the base Q on the template, it is unable further to extend probe A
since dQ'TP is not provided in the reaction mixture as dt~TP's. Extension terminates precisely at the point of ligation with the extended 3' end of probe A
abutting the 5' end of probe B. It is not known if the presence of probes A' and B on the template will terminate extension. But even in their absence, extension is terminated if attention is paid to proper stopbase selection.
Next, a ligase is employed to join the 3' hydroxyl end of extended probe A to the S' phosphate end of probe B to form a double stranded complex of fused or ligated primary probe and target strand. If the target is double stranded and has a complement T', the ligase will also join probes A' and B' in the initial cycle if they are hybridized to the target complement. If they are hybridized to excess probes A
and B
rather than target complement, ligation is inhibited since the ends are neither blunt nor sticky and there is no substrate for ligation.
Subsequently, the double stranded complexes are dissociated and new probes A, A', B and B' are permitted to hybridize to the target, the target complement, and both of the fused polynucleotides from the first cycle. Extension and ligation occur as befpre and the process can be repeated.
Some exemplary combinations of X's and Y's, their dNTP counterparts and the resultant possibilities for 0 and Q' are given in Table I.
..:..,_.... . ..,.:.... ., ,.
'VV~ 93/OD447 ~ ~ ~ ~ PCf/U~92/05A77 _1S-TABLE I
ILLUSTRATIVE GAP SEQUENCES, REDUIRED dNTPs, and POSSIBLE
COMBINATIONS FOR 0 and Q' IN DOUBLE GAP VARIATION
~n,L.C! YmLM X'TPs Y'TPs j.'~t and ~TO..P~AsE
A A T T T,C,G A,G,C
G 'f C A C, A G, T
AT AT T, A T,A C, G C, G
,r.,C C,e, T, G C,T T A
ATG AAA T, A, C T C G
G G C AAACG ~, G T,G,C T A
C
ATTGA AGGT T, A, C T,C,A C G
~ ~ G G C G complement.
C
not permitted The set not N,~ not M provides the possible complements (Q) for the stopbase D'.
The actual stopbase (Q') possibilities are given in the next column.
In tine case of double gap s, the length of gaps Xn and Ym may be one or any integer greater than one. For example, gyps of from t to 20 bases would be possible.
Practically, however, gaps of much shorter length are preferred; for example from 1 to 3 or S bases: It has been found that gaps of just one or two bases greatly increase the ratio of true signal t:o background and leave the largest number of options for stopbases and dXTP's. Since probes are actually designed around existing targets, 1 S rather than "selecting' stopbases, single or two base gaps are desirable in most cases.
Furthermore, it has been discovered that the double gap embodiment is preferred to the single gap version.
TARGET SEQUENCE CHARACTERlSTlCS
For clarity; double gaps are represented herein as "DG p,q" where "p" is the number of bases in the gap in one strand, and "4" is the number of bases in the gap of the other strand. Thus, d preferred double gap embodiment has two bases missing from each of the two probes whose 5' end participates in the ligation, and is designated DG 2,2. in this preferred embodiment, the 3° ends of the other two probes 25 do not overlap; rather they terminate at the same point on the target strand (and its complement). Also for clarity in this application the use of a dot or period "."
V1~~'l 9100447 PCT/~JS~2/05~~7 2~.~.~(.~~
between bases in a target sequence represents the point at which the probes, when hybridized to their complementary probes, would be blunt-ended but for the gaps in two of the probes. In other words, probes A and B' (see Fig. 3> have 3' termini which, absent the gaps in probes A' and B, would be blunt-ended at the point of , ligation. It is this point at which ligation would have occurred that is designated by a dot. However, with gaps present according to the invention, probes A and B' are extended, thus creating two, offset points of actual ligation.
In a particularly preferred DG embodiment bath the gaps are finable with just one type of deoxyribonucleotide triphosphate. This requires selecting a target sequence having the sequence -Ep.E'q-, where E represents any base, and p and q are integers between 1 and about 10. The probe sets must be designed so that the two probes whose ~' ends participate in the extension/ligatton (eg. probes A and B' tn Fig.
3) both terminate at the dot "." between E and E'. In order to provide a "stopbase" a further requirement is added. The target sequence must have a base other than E in 1 S the next position outward from the E string; and, similarly, must have a base other than E' in the next position outward from the E' string. This can be represented as -LEp.E'qJ-, where L is any base other than E and J is any base other than E'.
Final ly, to provide sufficient probe length for easy hybridization and, optionally, to provide specificity; the regions (N>h and (N)k are employed, to give:
5'-( N)hLEp.E'qJ(N)k-3' where N represents any base independently of any other base, N; and h and k are each integers greater than 5. Generally, to provide the desired specificity, the individual probes must be at least 10 or 1 1 nucleotides long, i.e., the sums (p*h> and (q*k) must be at least 10; more usually will be about 20 to 40, but can be up to 100 or more. As mentioned, p and q may be the same or different, and generally will be small, eg. 1-3.
In this preferred DC 2,2 system, probes would be designed and synthesized as follows; using the designations given in Fig. 3:
A' Hapten~--3'_(N')r,~L~_5' 3'-Eq.J'(N'>k~-5~°Hapten~ B' A Hapten~_5'_(N)h, L Eh_3' s'-J (N)~,t-5~-Hapten2 B
wherein the terms are defined as above except that h' and k' need not equal h and k exactly. It is entirely permissitrle for any of the probes to be slightly shorter or lohger than either the target sequehce or the complementary probe. This variation in length is indicated by the ""' symbol. The variation is usual ly no more tnan lu-25% of the probe length; preferably 10~ or less. Of course, no variation in length is also possible. Moreover, hand k' may represent a different number at each WO 9/00447 ~ ~ ~ ~ PCI'~US92/0~47~
occurrence, provided they are within the 20- 25~ variation limit; thus, probe A
need not be exactly p bases langer than probe A' (their (N)ht portions may wary in length too) and the same is true for k' and probes B and B'.
The single deoxyribonucleotide triphosphate of E' only is needed to fill all the S gaps. The bases L and J perform the same function as Q, i.e. to code for the stopbase Q'. However, designations L and J are used here because of the additional restrictions placed on them in this embodiment. As described elsewhere in this specification, haptens 1 and 2 serve to enable separation and detection of ligated products.
They are particularly useful for automated detection in the IMxo instrument (Abbott Laboratories). Napten 1 should be different than hapten 2. Virtually any low molecular weight compound to which antibodies can be raised may serve as hapten.
Several examples of this preferred embodiment are given in the first column of Table II below and in the Examples which fallow.
Another more generalized embodiment of the invention utilizes target sequences and probe sets wherein just two types.of deoxyribonucleotlde triphosphates are needed to fill all the gaps. Examples of this embodiment are given in the second ' column of Table I I and its target sequence is represented by the fallow~ng formula:
5"°(N)hXEp.FaZ(N)k-3, wherein N, p, q, h and k are all defined as before. E may be any base as before. F may be any base except E (if it were E the ends would be "sticky" when p=q), but when it is E' this case degenerates to the simpler embodiment described ,above.
Because of the additional flexibility in the choice of F, additional restrictions are placed on the stop bases X and Z. X may be any base but E or F', while Z may be any base but F or E'.
The gaps are filled by he deoxyribonucleatide triphosphates of E' and F.
z5 Ln this DG 2,2 system, probes would be designed ahd synthesized as follows, using the designations given ire Fig. 3:
A' Hapted~_3'_(N~)r~~~_5' 3'_F,aZ~(N'>~.t--5~-Napten2 B
A Hapteni_s'_(N)ht X Ep_3' y_Z (N)k9-s~~-Hapten2 B
The two deoxyribonucleotide triphosphates needed to fill all the gaps are F
and E'. The bases X and Z perform the same function as Q to cads for the stop base Q'.
However, designations X and Z are usbd here because of the additional restrictions placed on ahem in this embodiment. the restrictions are ~tso different than the restrictions on 35 L and J. The haptens are as described above:
Ct is important the "X" which codesfor a stopbase here (or X or X' used in a probe) should not be confused with the Xn gap or the dX'Tps needed to fail the Xn gap.
Regrettably, there are only twenty-six letters in the alphabet and most have been '~O 93/00447 PCTlUS92/05~''~
used in this application. It is believed that confusion between the two uses of X and X' is avoided by the context.
Other variations of this invention include DG configurations other than 2,2.
As shown in Table f I and in the examples, DG 1, t ; DG 1,2 (as well as 2, t ); DG 2,3 S (as well as 3,2> and DG 3,3 are also possible. In addition, gaps of up to 10 bases are included in the generic p and q case, although Table I I I below shows the low probability of gap junctions longer than about 3. 'There may be some advantage to having p be approximately equal to q (i.e. varying by only 1 or 2), but this is not a strict requirement. Though not tested, there is no reason to believe a double gap of 1,3 or 2,4 will not also work in the invention.
The above discussion describes in detail the "Special " symbois used in Table II. The "Conventional" symbols will not need further explanation to those of ordinary skill in the art.
TABLE II
~t 5 EXEMPLARY SINGLE and TWO dNTP FILL
DOUBLE GAP ("DG") JUNCTIONS
(The dot "."serves only to align sequences in the Table and to divide between right and left probe sets: All targets are written with their 5' end to the left. ) Gaps finable with just one Gaps fillable with just two deoxyribonucleotide deoxyribonucleotide triphosphate type triphosphates types - ~N)hLE.E'J(N)k (N>hXE,FZ(N)k DG 1,1 Generic; E' fills eneric cases)E' & F fill generic cases) (in all,~ (in all Subgeneric: (N)r,DC.GH(N)kG fills (N)hKC.TM(N)kG&T fill (N)r,HG.GD(N>kC fills (N>hYG.TR(N)kC&T fill (N>r,BA.TV(N)kT fills (N>r,RC.AY(N>kG&A fiil (N)hVT.AB(N)kA fills (N>hMG.AK(N)~C&A fill V~'~ 93100487 . ~ 1'C°if/~JS92/05477 DG 1,2,and 2,1 (N>hLE.E'E'JtN)k (N>hXE.FFZ(N>k Subgeneric: (N)hDC.GGH(N)k G fills(N)hKC.TTM(N)k G&T fill (N)hl-iG.CCDtN)k C tN)hKCC.TMtN)k G&T fill fills (N)hBA.TTV(N)k T fills(N)hYG.TTR(N)k C8~T fill tN)hVT.AAB(N)k A fills(N)hYGG.TR(N)~ CST fill (N)hRC.AAY(N)k G&A fill .
tN)hRCC.AY(N)k G&A fill (N)hMG.AAKtN)~ C&A fill (N)hMGG,AK(N)k C&A fill DG 2 2 Generic: tN)hLEE.E'E'JCN)k (N>hXEE.FFZtN)k Subgeneric: (N>hDCC.GGt-ltN)k G tN)hKCC.TTMtN)kG&T till fills (N)hHGG.CCDtN)k C fills(N)hYGG.TTR(N)kC&T fill , ~.:' tN)hBAA.TTVtN)k T fillstN)hRCC.AAYtN)kG&A fill tN)hVTT.AAB(N)~ A fillstN)hMGG.AAK(N>kC&A fill DG 2 3 and 3 2 (N)hLEE.E'E'E'J(N)k tN>hXEE.FFFZtN)k SubgeneriC: tN>hDCCC.GGH(N>k G (N)hKCCC.TTM(N)kGB~T fill fills (N>hHGGG.CCDtN)k C (N)hKCC.TTTM(N)kG&T fill fills tN)hBAAA.TTV(N>k T (N)hYGGG.TTR(N)kC8~T fill fills tN>hVTTT.AABtN)k A (N>hYGG.TTTR(N)kC&T fill fills (N)hRCCC.AAYtN)kG&A fill (N)hRCC.AAAYtN)kG&A fill (N>hMGGG.AAK(N)kC&A fill (N)r,MGG.AAAK(N)kC&A fill Generic: (N)hLEEE.E'E'E'JtN)k (N)hXEEE.FFFZ(N)k DG 3,~
__ (N)r,DCCC.GGGH(N)k (N)hKCCC.TTTM(N)~, Subgeneric: G fills G&T ffll (N)hHGGG.CCCD(N)~ C (N)hYGGG.TTTR(N)k fills C&T fill tN>~,BAAA.TTTV(N)k (N)nRCCC.AAAY(N)~;
T fills G&A fill tN)hVTTT.AAABtN>k A (N)hMGGG.AAAK(N)k fills C&A fill DG p,a (N)r,LEp.E'qJtN)k (N)hXEp.FaZtN)k GENERAL CASE E' alone fills E' and F alone fill Vl~~'~3/~dD~t7 ~ : : P(.'T/US~32/05~"''~
where the symbols have the following meanings:
CONVENTIONAL SPECIAL
A Adenine E any base .
B any base. but not A) F any base adenine C Cytosine L any base except E
D any base but cytosin(not J any base except E' C) G Guanine X any base except E or F' H any base but guaninenot G> Z any base except F or E' K G or T!U only h, any integer greater than i k M A or C only p, any integer between 1 and q N any base about 10 R A or G only S C or G only T Thymine U Uracil V any base but thymine/uracil w A or T/U only Y C or T/U onlv It will be understood that those of ordinary skill in the art will know haw to search for and identify specific target sequences meeting the requirements outlined above. For example many databases contain sequence information (eg, GENBANK, NBRF, EMBL or Swiss-Prot) which can be searched by readily available computer software (eg. MacVector, MacMolly or Geneworks>. It will also be realized that in any organism's known genome, multiple locations meeting the requirements will generally be found. For example, searching just the SF2CG isolate of HIV t (see Examples 1-S>, which contains approximately 9.7 kiiobases, reveals over 2000 possible DG locatiohs. The breakdown for each type of gap and fill situation is given in Table III. It should be realized that when p=q, a DG p,q on one strand at one location also comprises a DG p,q site an the opposite strand. In the situation where F=E' (ie. a single dNTP fill) the two target sites ace really just one location. Thus, for all 1 S symmetric double gaps twhere p~q) the Table reports the number actually found ' by searching. In contrast; an asymmetric DG (eg, a DG 1,2 or DG 2,3) on one strand represents a second, different site (but DG 2;1 or DG 3,2, this time) on the opposite strand. Thus, asymmetric sites are reported in the Table without halving the number.
W~ 93!00447 ~ ~ 1 ~'~ ~ l PC,')f/1<JS92/OS477 TABLE I l l Representative Numbers of Gaps by type in an Organism's Genomic DNA
Gaps finable with just Gaps finable with just Type of GAP one deoxyri;bo- two deoxyriba-nucleotlde nucleotide triphosphate type triphosphates types H1V t (SF 2CG Isotate)1 (9.7 kb) pG 1 1 1053 1099 e. .tr~act~~rn~t~s MOMP2 (2.2 kt?) ~ p . pG i 2 120 136 _____._._______ _ _. ._..__ 24 ........................_.... i 8 . ____~~_ ~.2 _._,-....,........
C. trachomat~s cryptic plastriid3 (7.5 kb) DG 1 2 4 i 5 488 pG 2.~,...~.-.... 61 55 aG 2 3 39 24 DG 3,3 6 6 , t: Sanchez-Pescado; R., et a1. Science 22'7:484-492 ( 1985), see examples t-5.
2. Baehr, W: et at: Proc.
Natl. Acad. Sct. USA 85:4000-4004 ( 1988) (EMBL Genbank Data Base: lnteiltgenetics, Accession ~J038131, see examples 1 1-12.
3_ Matt, C. et al. Nub. Acids Res. 16:4053-4067 (1988), see example 13.
For the HIV 1 isolate, thirteen of the 75 DG 2,2 single dNTP
fill sites are located within the gag region. These 13 locations awe listed in Appendix A.
In cases where genamic sequences are not known, at least certain portions must be established prior to using any version of LCR.
Typically, one of ordinary 15 skid can determine a sequence through routine methods, such as cloning and sequencing as taught in conventional textbooks on the subject, such as Maniatis, T, et ai. 'Molecular Cloning: A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor; NY (1982 and 1989>.
Whether one is seeking a universal probe set for several types, strains, or 20 serovars, etc. (as might be the case for HIV or Chlamydia?
ar one is seeking a f . _ , ._ ,:
'W~ 93100447 P~1'lt.3S92/05~~7 specific probe set to identify just one type (as might be the case for HPV), one of ordinary skill will know how to compare sequences obtained from such a search with the genomes of other organisms to identify conserved or unique sequences, respectively. In addition, routine laboratory investigations may be needed to confirm the universality or specificity of a particular probe set. The length and nature of tN)h and (N)k may greatly affect the universality or specificity of the respective probes, although it is also possible to design probes such that specificity is gained by the gap regions.
E~a~~~h~~ '.ores In a variation of either "recessed" embodiment, the deoxyribonucleotide triphosphates used in filling the gaps may be modified to contain a marker moiety.
Exemplary markers include direct labels such as radioisotopes, or hooks such as biotin, digoxin or other hapten capable of being recognized by either a solid phase or a 1 S label producing system. Isotopic labels include 32P, and 3H among others.
Incorporation of the marker into the dNTP's is generally a matter of conventional organic chemistry: Linkers or spacers may be used but are not essential. It is only important that the modified dNTP be able to be incorporated into the gap opposite its complement on the target strand and be covalently bonded to the adjacent base.
The invention will now be described further by way of examples which are illustrative of the invention and are not intended to limit it in any way. For example, sequences of specific length are listed. ft should be understood that sequences covering the same map positions but having slightly fewer or greater numbers of bases are deemed to be equivalents of these sequences and fall within the scope of the invention, provided they V~ill hybridize to the same positions on the target as the listed sequences.
Unless otherwise indicated, sequences in the examples are written with the 5' end toward the left. In the examples, some probes are labeled with haptens:
'°FL"
represents a fluorescein hapten and "BIO" represents a biotin hapten, covalently attached to the probe. The haptens are attached at either the 3' or 5' terminus as shown. Throughout the examples, the numbering system for probes corresponds to a map position followed by a decimal .i, .2, .3, or .4, which correspond to probes A, A', B, B', respectively, of Fig. 3. Thus, arranging the probes as: .1 : .3 .2 : .4 W~ 9310(D4~47 ~ ~ ~ ~ ~ ~ ~ P~'1'/1JS92/(D~477 yields an arrangement which is upside-down when compared to Fig. 2 or 3. The map position given is for the left-most base when the probes are aligned as if hybridized.
The remaining map positions can be calculated from this information, knowing that the left-most position bears the lowest number.
In most of the examples, results were read in an IMxo instrument. This is commericaily available from Abbott Laboratories and is described in EP-A-288 and ~in Fiore, M. et al Clin. Chem., 34/9:1726-1732 ( 1988). It should be noted that the IMx instrument typically generates Nmachine" noise or background in the range of 2-8 counts/sec/sec.
Quantities of polymerise are expressed in units, defined (eg., by MBR) as follows: 1 unit of enzyme equals the amount of enzyme required to incorporate nanomoles total nucleotides into acid-insoluble material in 30 min at 70'C.
lj:nits of ligase enzyme are defined (internally by Abbott Laboratories) as: t mg of 9S~
,, ~. ~ purified Thermus thermvphilus DNA ligase has a specific activity of about 1 x 1 Oa t S units. While this is not precisely standardized and may vary by as much as 20~, optimization is wi~hin the skill of the routine practitioner.
Examples 1-12 relate to detection of Human Immunodeficiency Virus (HIV1) , using several different sequences. The sequences, their map position (SF2CG
isolate according to Sanchez-Pescado, R., et al. Science 227:481-~i92 ( 1985)), and their Sequence ID Nos. are given in Table IV, below. Appendix A gives the number and map positions of DG2,2 targets in the gag region of HIV 1.
Table I V.
1667. t : FL-AACCCTTTAG AGACTATGTA GACC
1667.2: TCTACATAGT CTCTAAAGGG TTC-FL 2 1667.3: TTCTATAAAA CTCTAAGAGC CGA-BIO 3 1667.4: BID-CGGCTCTTAG AGTTTTATAG AACC 4 912.1: FL-AGAACGATTC GCAGTCAATC S
CTGG
912.2: AGGATTGACT GCGAATCGTT CTA-FL6 3S 912.3: TGTTAGAAAC ATCAGAAGGC TGC-BIO7 912.4: B10-CAGCCTTCTG ATGTTTCTAA 8 CAGG
2086. t : FL-GCTAATTTTT TAGGGAAGAT 9 CTGG
2086:2: AGATCTTCCC TAAAAAATTA GCC-FL10 2086.3: TTCCTACAAG GGAACGCCAG GGA-Bf01 2086.4: B10-CCCTGGCGTT CCCTTGTAGG i MoD ~93/~~447 ~ ~ ~ ~ ~ ~ ~ PCT/~J~9210~~'''7 789. t : FL-GATGGGTGCG AGAGCGTCGG TATT t 3 789.2: TACCGACGCT CTCGCACCCATCT-FL 14 789.3: GCGGGGGAGA ATTAGATAAA TGG-BIO 15 789.4: B10-CATTTATCTA ATTCTCCCCC GCTT 16 S
t5 508,1: FL-ACCCACTGCT TAAGCCTCAA TAAAG 17 ..
508.2: T1'TATTGAGG CTTAAGCAGT GGGTT-FL 18 508,3: TTGCCTTGAG TGCTTCAAGT AGTGT-BIO t 9 508.4: BIO-CACTACTTGA AGCACTCAAG GCAAG 20 , 5569. t : FL-GAACAAGCCC CAGAAGACCA AGGG ?_ 1 5569.2: TTGGTCTTCT GGGGCTTGTT C-FL 22 5569.3: ACAGAGGGAG CCATACAATG AA-BIO 23 5569.4: BIO-TTCATTGTAT GGCTCCC'TCT GTGG 24 t 450, t : FL--GCATGCAGGG CCTATTGCAC 25 CAGE
1450.2: TGGTGCAATA GGCCCTGCAT G-FL 26 t 450.3: AAATGAGAGA ACCAAGGGGA AG-BIO27 t 450.4: B10-ACTTCCCCTT GGTTCTC'TCA 28 TTTGG
t 5?3. t : FL-AGAAATCTAT AAAAGATGGA 29 THAT
t 573.2: TTATCCATCT.TTTATAGATT TCT-FL30 t 573.3: TGGGATfAAATAAAATAGTAAG-BIO 31 t 573.4: B10-CTTACTATTT T~TTTAATCC 32 CAGG
4060.1: FL-GCATTAGGAA TCATTCAAGC 33 ACAA
4060.2: GTGCTTGAAT GATTCCTAAT GC-FL 34 4060.3: AGATAAGAGT GAATCAGAGT TA-Bi035 4060:4: BIO-TAAC-fCTGAT TCACTCTTAT 36 CTGG
2683. t : FL-AAGGAAGGGA AAATTTCAAA 37 AATT
2683.2: TTTTTGAAAT TTTCCCTTCC TT-FL 38 2683.3: CCTGAAAA'TC CATACAATAC T-B1039 2683.4: Bi0-AGTATTGTAT GGATTTTCAG 40 GCCC
1753. t : FL--AACCTTGTTG GTCCAAAATG 4 CAAA t 1753.2: GCATTTTGGA CCAACAAGGT: f-FL 42 1753.3: AGATTGTAAG ACTATTTTAA A-BIO 43 t 753.4: Bl0-TTTAAAATAG TCTTACAATC 44 TGGG
3697. t : FL-GTAATATGGG GAAAGACTCC'TAAA45 3697.2: AGGAGTCTTT CCCCATATTA C-FL 46 3697.3: AAACTACCCATACAAAAGGAA-BI0 47 3697.4: B10-TTCCTTTTGT ATGGGTAGTT 48 TAAA
Double gap LCR (DG2,2) was performed for 40 cycles consisting of a 65 second incubation at 85°C and a 65 second incubation at 50°C. Reactions were set up with either 0 or t 02 target molecules. The target DNA was a BamH 1 fragment of HI
V t DNA
cloned into pBR322. Negative reactions contained t 0 nanograms of human placental DNA. Rositive reactions contained the indicated number of copies of HI V 1 plasmid DNA
in a background of t 0 nanograms of placental DNA. The LCR oligonucleotides used are listed in Table IV. These oligonucleotides are specific for the map positions t667-woo 9~roo~~ ~ ~ ~ ~ t~°~.~:. ~creus92rosa7~
t 716 within the gag region of the HIV1 genome (HIV SF2CG isolate, see Sanchez-Pescado>. Reactions were run in a buffer containing SOmM EPPS pH 7.8, 100 mM
KCI, 10 mM MgCl2, t mM DTT, 10 mM NH4C1, 100 uM NAD, 10 ug/ml BSA, 5 x 101 1 molecules each oligonucleotides 1667.1, 1667.2, 1667.3, 1667.4 (ID Nos.
S 1-4, respectively>, 1 uM 2'-deoxyguanosine S'-triphosphate, 0.5 units Thermos DNA polymerase (Molecular Biology Resources, Inc., "MBR">, and 3400 units Thermos thsrmophilus DNA ligase. Reaction volume Was SO pl and each reaction was overlaid with 25 ut of mineral oil prior to cycling.
Following amplification, reactions were diluted t:t with IMx di)uent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Num~r of ar,~t molecules $
0 S.?6 102 1686.8 Double gap LCR (DG2,2) was performed for 40 cycles using the same cycling parameters described in Example 1. Reactions were set up in duplicate with either 0, 10 or 102 target DNA molecules. Negative reactions contained 1 microgram of human placental DNA. Positive reactions contained 102 HIV target DNA molecules in 1 microgram of human placental DNA. The oligonucleotides used are listed ~n r ame ~v.
These oligonucleotfd~s are specific for map positions 9 i 2-961 within the gag region of the H(V 1 genome (HIV SF2CG Isolate>. Reactions were run in a buffer containing 50 mM EPPS pH7.8, 20 mM K*; 30 mM MgCl2, 7.5 x 101 1 molecules each oligonucleotides 912.1, 912.2;912.3,912.4 (lD Nos. 5-8, respectively), 100 pM
NAD, 1 pM 2'-deoxycytidine 5'-triphosphate, 0.5 units of Thermos DNA
polymerase (MBR), and 3400, units Therrrus th~rmophilus DNA ligase. Reaction volume was ~l and each reaction was overlaid with 25 t~l of mineral oil.
Following amplification; reactions were diluted 1:1 with IMx diluent buffer and amplification products were detected via a sandwich immunoassay performed with the Abbott IMx automated immunoa~sa~ system.
N~ b . r ~~t 1'0~~
0 3.8 1 Q 32.8 102 604.4 W(J 93/0~447 ~. PCT/US92/0. 7 Double gap LCR (DG2,2) Was performed for 40 cycles consisting of a 65 second incubation at 85°C and a 65 second incubation at 60'C. Reactions were set up in quadruplicate with either 0 or 102 HIV 1 target DNA molecules. Negative reactions contained 1 microgram of human placental DNA. Positive reactions contained 102 HIV
1 target DNA molecules in l microgram of human placental DNA. The ollgonucteotides used are listed in Table IV. These oligonucleotides are specific far map positions 2086-2135 within the gag region of the HIV 1 genome tHIV SF2CG isolate) except that the nucleotide in position 15 of oligonucleotide 20863 was changed from G
to C
to disrupt a possible intramolecular hairpin structure tposition 8 in oligonucteotide 2086.4 changed from C to G). Reactions were run in a buffer identical to that used in example 2 except that oligonucleotides 2086.1, 2086.2, 2086.3, and 2086.4 tID
Nos. 9-12, respectively) were used at 7.5 x 101 1 mol,ecutes each per reaction.
Following amplification; r~eactians were diluted 1:1 with IMx dituent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~i~llb~t of ar~et m_.plecules 0 5.9 , 02 249.5 Exai lie 4.
Doable gap LCR tDG2;2) was performed for 40 cycles using cycling parameters identical to those used in Example 3. Reactions were set up quadruplicate with either O or 102 HIV l target DNA rnolecules. Negative reactions contained 1 microgram of human placental DNA, Positive reactions contained 102 HIV 1 target DNA
molecules in 1 microgram of human placental DNA. The oligonucleotides used are listed in Table IV. These oligonucleotidesare'specific far map position 789-838 within the gag region of'the HI V 1 gen~me tHl V SF2CG isolate?. Reaction conditions were identical to those in Example 2 except that oligohuc)eofides 789.1, 789.2, 789.3, and 789.4 at 7.5 x 101 1 each ( ID Nos. l 3- t 6, respectively), and 2-deoxyadenosine 5'-triphosphate at 1 ~M were used, Following amplification, reactions were diluted i:i with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
j~,~ber of tart molecules $~rg 0 3.8 p2 604.4 W~ 93/~D044? ~ ~ ~ ~ ~ ~ PCT/US92/0547?
Double gap LCR tDGl,1> was performed for 45 cycles consisting of a 6S second incubation at 85°C and a 65 second incubation at 65'C. Reactions were set up in duplicate with~either 0 or 102 target DNA molecules. Negative reactions contained nanograms of human placental DNA. Positive reactions contained 102 HIV 1 target DNA molecules in 10 nanograms of human placental DNA. The oligonucleotides used are specific for map posttions 508-559 within the LTR region of the HIV 1 genome ' fHIV SF2CG isolate). Reactions were run in a buffer identical to that used in Example 10 1 except that oligonucleotides 508.1, 508.2, 508.3, and 508.4 tID Nos. 17-20, respectively) were used at 5 x i 011 molecules each and 2'-deoxycytidine S'-triphosphate was used at 1 uM.
Following amplification, reactfions were diluted 1:1 with IMx diiuent buffer, and the LCR amplification products wire detected v1a a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
L~,"~'~l<L ~~C9~.~olecules 0 4.5 102 1284.8 ~amnle 6.
Double gap LCR (DG2,3) is performed far 30-SO cycles as in the previous examples. The oiigonucleotides used are listed in Table IV, and are specific for map positions 5569-5516 within the vpu coding region of the HI V 1 genome (HI V
isolate): Reaction conditions are like those described in Examples 1 - 3, except that approximately 5 x 101 1 molecules each of otigonucleotides 5569.1, 5569.2, 5569.3, 5569.4 (ID Nos. 21-24; respectively, and approximately 1 p.M 2'-deoxycytadine S'-triphosphate are used.
Following amplification, the reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
JE,~ ~!~ l e?.
Double gap LCR (DG2,2) was performed for 4S cycles consisting of a 6S second incubation at 90°C and a 65 second incubation at 65'C. Reactions were set up in duplicate with either O or 102 HIV 1 target DNA molecules. Negative reactions contained 10 nanograms of human placental DNA. Positive reactions contained HIV 1 target molecules in 10 nanograms of placental DNA. The oligonucleotides used are listed in Table IV. These oligonucleotides are specific for map positions 1498 within the gag region of the HIV 1 genome (HIV SF2CG isolate). Reactions were run in a buffer identical to that described in Example 2 except that S x 101 1 molecules each of oligonucleotides 1450.1, 1450.2, 1450.3, and 1450.4 (ID Nos.
S 25-28, respectively) were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number of target molecules , Rate 0 4.8 102 1932.5 Double gap LCR (DG2,1: 2 dNTPs> is performed for 30-50 cycles as in the previous examples. The oligonucleotides used are listed in Table IV, and are specific for map positions 1573-1620 within the gag region of the HIV 1 genome tHIV
isolate). Reaction conditions are similar to those described in Examples 1 -3, except that approximately 5 x 101 1 molecules each of oligonucleotides 1573.1, 1573.2, 1573.3, 1573.4 (ID Nos. 29-32, respectively), and approximately 1 uM each 2'-deoxycytidine S'-triphosphate and 2'-deoxyadenosine 5'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Double gap LCR (DG2,2: 2 dNTPs) are performed for 30-50 cycles as in the previous examples. The oligonucleotides used are Listed in Table IV, and are specific for map positions 4060-4107 within the poi region of the HIV i genome (HIV
isolate). Reaction conditions are similar to those described in Examples 1 -3, except that approximately S x 101 1 molecules each of oligonucteotides 4060.1, 4060.2, 4060.3, 4060.4, LID Nos. 33-36, respectively) and approximately 1 uM each 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate are used.
Following amplification, reaction are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~o ~~iooaa~ 2 ,~ ~, ~ ~ ~ ~ Pcrius9z'o~a7~
Double gap LCR EDG3,2:2 dNTPs) is performed for 30-SO cycles as in the previous examples. The oligonucleotides used are listed in Table IV, and are specific for map positions 2683-2730 within the gag region of the HIV 1 genome (HIV
isolate). Reaction conditions are similar to those described in Examples t -3, except that approximately S x 10 i > molecules each of oligonucleotides 2683. ! , 2683.2, 2683.3, 2683.4 (ID Nos. 37-40, respectively>, and approximately 1 uM each 2'-deoxyadenosine 5'-triphosphate and 2'-deoxyguanosine S'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with Ihlx diluent buffer and amplification products are detected vfa a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~Q~le 11.
Double gap LCR (DG3,3:2dNTPs) is performed for 30-SO cycles using the t S oligonucteotides listed in Table IV. These oligonucleotides are specific for map positions f 753-t 800 within the gag region of the HIV 1 genome (HIV SF2CG
isolate>. Reaction conditions are similar to those described in Examples t -3, except that approximately 5 x 10i 1 molecules each of oligonucleQtides 1753.1, 1753.2, 1753.3, 1753.4 tID Nas. 41-44, respectively), and approximately 1 ~M 2'-deoxythymidine S'-triphosphate and 2'-deoxycytidine 5'-triphosphate are used.
Following amplification, reaction is diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
~=xa 1~I?_.
Double gap LCR (DG3,3> is performed for 30-SO cycles as in the previous examples. The oliganuGieotides used are listed in Table IV, and are specific for map positions 3697-3744 w itf,in the poi region of the HI V t genome (Hi V t SF2CG
isolate) except that the nucleotide in position 17 of oligonucleotide 3697. i was changed from T to C to disrupt a possible intramolecular hairpin structure (position S in oligonucleotide 3697.2 changed from A to G). Reaction conditions are like those described in Examples 1-3; except that approximately 5 x 101, molecules each oligohucleotides 3697:1, 3697.2, 3697.3, 3697.4 (' ~ Nos. 45-48, respectively>, and 1 pM 2'-deoxythymidine 5'-triphosphate are uses.
Following amplification, the reactions are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
A) ~~~." :',~, ' .. .. ;v:: '.~.:.
i~~ ~3>07 PC'1"/LJS32/05~?7 Examples 13-15 relate to the detection of HI V 2 DNA. The sequences, their map position according to Franchini, G., et a1. Proc. Natl. Acad Sci. 86:2433-( 1988>, and their Sequence 10 Nos. are given in Table V, below.
- ~ TABLE V
2060. t : FL-GACAGAGGAC TTGCTGCACC 49 TCAA
2060.2: GAGGTGCAGC AAGTCCTCTG TCG-FL50 2060.3: CTCTCTTTGG AAAAGACCAG TAG-BIO51 2060.4: BIO-TACTGGTCTT TTCCAAAGAG s2 AGAA
4661.1: FL-ATCACCACAG AACAAGAAAT 53 ACAA
4661.2: GTATTTCTTG TTCTGTGGTG ATC-FL54 4661.3: CCTCCAAGCA AAAAATTCAA AAT-BIO55 4661.4: Bi0-TTTTGAATTT TTTGCTTGGA 56 GGAA
1094.1: FL-CTATGATATT AATCAAATGC 57 TTAA
~ r ' 1094.2: AAGCATTTGA TTAATATCAT AGG-FL58 1094.3: GTGTGGGCGA CCATCAAGCA GCG-BIO59 1094.4: BIO-GCTGCTTGAT GGTCGCCCAC 60 ACAA
2S Double gap LCR tDG2,2> was performed for 45 cycles using cycling parameters identical to those described in Example 1. Reactions were set up in triplicate with either 0, 102, or 103 target molecules. The target DNA was a segment of HIV 2 genamic DNA cloned into pTZ 18. Negative reactions contained 1 microgram of human placental DNA. Ppsitive reactions contained the indicated number of copies of containing plasmid DNA in a background of 1 microgram of human placental DNA.
The oligonucleotides used are listed in Table V. These oligonucleotides are spdcific for map positions 2060-2109 within the gag region of the HIV 2 genome (I-IIV21 SY
isolate) except that the nucleotide in position 14 of oligonucleotide 2060.3 was changed from C to A to disrupt a possible intramolecular hairpin structure (position 9 in oligonucleotide 2060.4 changed from G to T>.: Reactions were run in a buffer identical to that described in Example 2 except that 5 x 101 1 molecules each oligonucleotides 2060.1, 2060.2, 2060.3, 2060.4 tiD Nos.49--52, respectively>, and 1 ~M 2'-deoxythymidine 5'-triphosphate were used.
Following amplification; reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected viva sandwich immunoassay performed using the Abbott I~lx automated immunoassay system.
°
W~ 93/i77 ~ ~. ~. ~ ~ s Number of target m~j$a~
0 .8.4 102 318.4 103 789.3 S
Double gap LCR (DG2,2> is performed for 30-50 cycles as in the previous examples. The oligonucleotides used are Listed in Table V, and are specific for map positions 1094-1 143 within the ga,greglon of the HIV 2 genome (HIV21SY
isolate).
Reaction conditions are like those described in Examples 1-3, except that approximately 5 x 101 1 molecules each oligonucleotides 1094.1, 1094.2, 1094.3, 1094.4 ( ID Nos. 53-56, respectively), and 1 pM 2'-deoxythymidine 5'-triphosphate are used.
Following amplification, the reactions are diluted 1:1 with IMx diiuent buffer and 1~~ ~ amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Double gap LCR (DG2,2> is performed for 30-50 cycles as in the previous examples. The oligohucleotides used are listed in Table V, and are specific for map positions 4661-4710 within the HIV 2 genome (HIV21SY isolate). Reaction conditions are like those described in Examples 1-3, except that approximately 5 x 101 1 molecules each oligonucleotides 4661.1, 4661.2, 4661.3, 4661.4 (IQ Nos.
57-60, respectively), and 1 pM 2'-deoxycthymidine 5'-triphosphate are used.
Following amplification, the reactions are diluted 1:1 with IMx diluent buffer and amplification products are detected via a sandwich immunoassay performed with the Abbott IMx automated immunoassay system.
Examples 16 - 19 relate to detection of Human Papilloma Virus (HPV), types 16 and 18. The sequences and their Sequence ID Nos are given in Table VI. Map positions for type 18 (Examples 8 and 10) are found in Seedorf, K., et al.
Virology 145:181-185 ( 1985); while map positions for type 16 are found in Zhang, Y.-X., Morrison, S.G., and H.D. Caldweli; Nuc. Acids Res. 18:1061 ( 1990>.
W~ 9?1/UU4~~7 ~ ~ ~ ~ ~ ~ P~ I'/1JB92/05~~7 Table W .
631.1: FL-TTTAGAGCCC CAAAATGAAA TTCC 61 631.2: AATTTCATTT TGGGGCTCTA AA-FL 62 631.3: TTGACCTTCT ATGTCACGAG -B10 63 631.4: BIOCTCGTGACAT AGAAGGTCAA CC 64 480.1: FL-ATAATATAAG GGGTCGGTGG ACC 65 480.2; TCCACCGACC CCTTATATTA T-FL 66 480.3; TCGATGTATG TCTTGTTGCA GA-B10 67 480.4; BIO-TCTGCAACAAGACATACATC GACC 68 488.1: FL-CAACATAGCT GGGCAC'fATA GAGG69 488.2. TCTATAGTGC CCAGCTATGT TG-FL 70 488.3; AGTGCCATTC GTGCTGCAAC C-B10 71 488.4; BIO-GGTTGCAGCA CGAATGGCAC TGG 72 6604.1 FL-TTTGTTGGGG TAATCAATTA TTTGTT73 ,: - ~ 6604.2 CAAATAATTG ATTACCCCAA CAAA-FL 74 6604.3 CTGTGGTTGATACCACACGC A-BIO '75 6604.4 B10-TGCGTGTGGT ATCAACCACA GT 76 Example 16.
Double gap LCR was performed for 35 cycles using the same cycling parameters described in Example 1. Reactions were set up in with either 0 or 102 target DNA
molecules. The target DNA was full length HPV 18 genomic DNA cloned into pGEM3.
Negative reactions contained 15 nanograms of calf thymus DNA. Positive reactions contained 102 HPV 18 target DNA molecules in l5 nanograms of calf thymus DNA.
The oligonucieotides used are listed in Table Vl. These oligonucleotides are specific for map positions 631-676 in the E6/E7 region of the I~iPV 18 genome 3.
Reactions were run in a buffer identical to that described in Example 1 except that 5 x molecules each oiigonucleotides 631.1, 631.2, 631.3, 631.4 (ID Nos.
61°64, respectively) and t pM 2'-deoxyguanosine 5'-triphosphate were used.
Following ampltficatioh, reactions were diluted t:1 with IMx diiuent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
1~ t~L ~ e~Qlecules B~, 0 6.8 1 p2 ~ 166.6 WO 93/UiD447 , PCT/LJS92/OS477 ~1~.~~2 Fxam~le 17.
Double gap LCR (DG2,2) was performed for 35 cycles using reaction conditions .
identical to those described in Example 3. Reactions were set up in with either 0 or 102 target DNA molecules. The target DNA was full length HPV i 6 genomic DNA
cloned into pSP65. Negative reactions contained 15 nanograms of calf thymus DNA.
Positive reactions contained 102 target HPV 16 DNA molecules in 15 nanograms of calf thymus DNA. The oligonucleotides used are listed in Table VI, These oligonucleotides are specific for map positions 480-526 within the E6/E7 region of the t-IPV 16 genome. Reactions were run in a buffer identical to that used in Example 1 except that 5 x 101 1 molecules each oligonucleotides 480.1, 480.2, 480.3, 480.4 (iD Nos. 65-68, respectively>, and 1 uM 2'-deoxyguanosine 5'-triphosphate were used.
Fot,lowing amplification; reactions were diluted 1:1, with IMx diluent buffer, and ,, r ~ the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
N~!f bn ~r of target JCtt~~g ~~ fi~
0 5.9 102 28.6 E,~.m~ 1 a ~ 8.
Double gap LCR tDG2,2> was performed for 35 cycles using the same cycling parameters described in Example 1. Reactions were set up in duplicate with either 0 or 106 target DNA molecules. The target DNA was full length HPV 18 genomic DNA
cloned into a plasmid: Negative reactions contained 15 nanograms of calf thymus DNA.
Positive reactions contained 106 target HPV 18 DNA molecules in 15 nanograms of calf thymus DNA. The oligonucieotides used are listed in Table VI. These oligonucleotides are specific for map positions 488-534 within the E6/E7 region of the HPV 18 genome. Reactions were run in a buffer identical to that described in Example 1 except that 5 x t011 molecules each otigonucleotides 488.1, 488.2, 488:3, 488.4 tID Nos. 69-72, respectively), and 1 uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification; reactions were diluted 1:1 with if1x diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
VVCD 9310047 ~ ~ ~ ~, ~ ~ ~ PC'~'l~JS92/OS~''~
_34_ b.r of ~~~et molecules p 7.0 106 978.2 ~xam~~ 19.
. Double gap LCR tDGl,2) was performed for 40 cycles consisting of a 65 second incubation at 88'C and an 80 second incubation at 50'C. Reactions were set up in triplicate with either 4, 104, or 106 target molecules. The target DNA was full length viral genomes cloned into various plasmids. Alt reactions contained 330 nanograms of human placental DNA. Positive reactions contained 104 or 106 molecules of NPV 6, 16, 18, 31, 33, or 61. The oligonucleotides used are listed in Table VI. These oiigonucleotides correspond to map positions 6604-6651 in the HPV
16 genome, but do not match this sequence exactly. Nucleotide changes representing ~.:- ~ consensus between all of the HPV types listed above were introduced during synthesis. .
Reactions were run in a buffer identical to that described in Example 2 except that 4 x 1012 molecules each of oligonucleotides 6604.1, 6604.2, 6604.3, 6604.4 tID
Nos. 73-76 respectively), i 8;000 units of Thermos thermaphilus DNA ligase, 4 units of Thermos DNA polymerise (MBR); 1 OuM NAD, and l.7uM 2'-deoxyadenosine S'-triphosphate were used. Reaction volume was 200 ul with no mineral oil overlay.
Following amplification, LCR reaction products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Rate tc/s/s) tai ~ ~, ~ .~lP.~ t.~. tl~l molecules Tvae 66 Tvae L~ TYQe 18 ~~.J.. .LYE-.~ ~.1 0 ~ 6.6 6.6 6.6 6.6 6.6 6.6 104 ~ 355.4 31.4 15.3 66.9 78.8 7.4 1 Ob ~ 2 t 73. 7 1 163.8 805.5 1439.7 1597.8 62.1 Examples 20 - 21 relate to detection of ~ierpes Simplex Virus tf~SV). The sequences used; their map position according to McGeoch; D.J. et al. J. Gen.
Virol.
68;19-38 t 1987>, and their Sequence ID Nos. are given in Table V.
~c~ ~~i~7 ~ ~. ~ ~ ~ ~ ) >P~ri~~9zaosa~~
Table V! I.
475 ) .1; FL-CCCCCTGTTC TGGTTCCTAA 77 CGG
475 ) .2: GTTAGGAACC AGAACAGTGG GGA-FL.78 475 ) .3: TCCCCTGCTC TAGATATCCT CT-BIO79 475 ) ,4: BIO-GAGGATATCT AGAGCAGGGG 80 AGG
0 6465. ) : F!_-TATGACAGCT TTAGCGCCGT 8 i CAG
6465.2: TGACGGCGCT AAAGCTGCAT AG-Fi"82 6465.3: GAGGATAACC TGGGGTTCCT GAT-BIO83 6465.4: BIO-TCAGGAACCC CAGGTTATCC 84 TCG
i 5 ~"~t1!~ n 1 a 2 0.
Double gap LCR (DG2,2) was performed for 60 cycles consisting of a 60 second incubation at 85°C and a 60 second incubation at 50°C. Reactions were set up in duplicate with either 0 or ) 02 target DNA molecules. The target DNA was a 4538 by ~ f , segment of the HSV 2 genome cloned into pUC ) 9. Negative reactions contained 10 20 nanograms of human placental DNA. Positive reactions contained 102 H5V 2 plasmid molecules in ) 0 nar~ograms of human placental DNA. The otigonucleotides used are listed in Table VII. These oligonucleotides are specific for map positions 475)-4798 within the US4 region of the NSV 2 genome 2. Reactions were run in a buffer identical to that described in Example ) except that oligonucleotides 475).1, 475).2, 25 4?51.3, and 475 ) .4 ( i D Nos. 77-80, respectively) at 5 x ) 0 ~ ) molecules each per reaction, and ) uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted ):) with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay perfarmed using the Abbott IMx automated immunoassay system.
r of t~C eat mold .
p 6.0 102 2096.0 Double gap LCR (DG ) ,1 ) was performed for 60 cycles using cycling parameters identical to those described in Example t. Reactions were set up in duplicate with either 0 or 102 target DNA molecules. Negative reactions contained 10 nanagrams of human placental DNA. Positive reactions contained ) 02 HSV target DNA
mo)ecules in q0 ) 0 nanograms of human placental DNA. Tlie o)igonucleotides used are listed in Table V19. These oligonucleotides are specific for map positions 6465-65)2 at the genome. Reactions were run in a buffer identical to that described in Example ) except that oligonucleotides 6465.1, 6465.2, 6465.3, and 6465.4 (ID Nos. 81-84, respectively) at 5 x 101 1 each per reaction and 1 uM 2'-deoxycytidine 5'- .
triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx dituent buffer, and LCR reaction products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number o~ target mQiecules $~g 0 6.0 102 1 156.0 Examples 22 - 24 relate to detection of Chlamydia trachomatis DNA. The sequences used and their Sequence ID Nos. are given in Table VI I. The map positions for the MOMP region (Examples 1 1 and 12) are according to 8aehr, W. et al.
Proc.
Nati. Acad. Sci. USA 85:4000-4004 ( 1988) fEMBL Genbank Data Base:
Intelligenetics, Accession *J03813]; while the map positions for the cryptic plasmid region (Example 13) are according to Hatt, C. et a1. Nuc. Acids Res.
16:4053-4067 ( 1988).
Table VIII.
36. i : FL-TTTTACTTGC AAGACATTCC TCAGG85 36.2: TGAGGAATGT CTTGCAAGTA AAAGC-FL86 36.3: ATTAATTGCT ACAGGACATC TTGTC-BIO87 36.4: BIO-CAAGATGTCC TGTAGCAATT ~
552.1: GGGAATCCTG CTGAACCAAG 89 552.2: TTGGTTCAGC AGGATTCCC 90 552.3: TTATGATCGA CGGAATTCTG TG 91 552.4: CACAGAATTC CGTCGATCAT AAGG , ~2 6693.1: FL-GATACTTCGC ATCATGTGTT CC 93 6693.2: AACACATGAT GCGAAGTATC -FL 94 6693.3: AGTTTCTTTG TCCTCCTATA ACG-BIO 95 6693.4: BIO-CGTTATAGGA GGACAAAGAA ACTCC 96 Double gap LCR (DG2,2) was performed for 35 cycles using cycling parameters identical to those described in Example 3. Reactions were set up in duplicate with either 0 or 102 target DNA molecules. The target DNA was purified Chlamydia trachomatis genomic DNA. Negative reactions contained 330 nanograms of human placental DNA. Positive reactions contained 102 Chlamydia target DNA molecules in * Trade-mark ~4 93/00447 '~ ~ ~ ~" ~ ~ ~ ~ ~ PC'T/U~92105477 330 nanograms of human placental DNA. The oligonucieotides used are listed in Table Vlll. These oliganucleotides are specific for map positions 36-89 within the major outer membrane protein (MOMP) gene. Reactions were run in a buffer identical to that described in Example 2 except that 5 x 101 1 molecules each oligonucleotides S 36.1, 36.2, 36.3, 36.4 tID Nos. 85-88. respectively) and t uM 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with lMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
1a ~mne of a~~mol .ec~les .B~t,~.
0 7.2 102 34.2 15 EXa l~ a 23.
Double gap LCR (DG 1,2) was performed for 40 cycles consisting of a 60 second incubation at 85°C and a 60 second ihcubation at 59'C. Reactions were set up in dqplicate with either O or 102 target molecules. Negative reactions contained nanograms of human placental DNA. Positive reactions contained i 02 Chlamydia 20 target DNA molecules in 330 hanograms of human placental DNA. The oligonucteotides used are listed ire Table Vll I. These otigonucleotides are specific for map posftions 552-595 within the MOMR gene. Reactions were run in a buffer identical to that described in Example 3 except that 8 x 101 1 molecules each of oligonucieotides 552.1 552.2 552.3 552.4 (ia Nos. 89-92, respectively), and 1 >aM oc 32P
labelled 25 ' 2'-deoxycytidihe 5'-triphosphate are used.
Following amplification, the reaction products were separated by electrophoresis on a 10% polyacrylamide gel with B.OM urea at 330 v constant for about 2 hours.
The gels were then autoradiog~aphed for about l2 hours with intensifying screens toptionaT>. In the presence of target; autoradiography revealed bands equitable with 30 the extended, unligated probe, and the longer, ligated product. The negative control gave no discernible bands.
. ExamQle 4.
Double gap LCR (D~2,~) was performed for 40 cycles consisting of a 60 second 35 incubation at 85'C and a 60 second incubation at 59'C. Reactions were set up in duplicate with either O or 102 target'molecules. Negative reactions Contained nanograms of human placental DNA. Positive reactions contained 102 Chlamydia target DNA molecules in 330 nanograms of human placental DNA. The oligonucteotides v~o ~~~oo~~ ~~ -~ 1 ; n ~ :~ ~cr~us9zios~~~
_ :. ,.'i ;~
used are listed in Table VII I. These oligonucleotides are specific for map positions 6693-6739 within the Chlamydia trachomatis cryptic plasmid. Reactions were run in a buffer identical to that described in Example 3 except that 8 x 1011 molecules each oligonucleotides 6693.1, 6693.2, 6693.3, 6693.4 (ID Nos. 93-96 S respectively>, and 1 ~M 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott iMx automated immunoassay system.
Number o~"~Lg~t mote~u_le~ 8~
0 4.8 102 1538.8 Examples 25 - 29 relate to detection of Neisseria ~onorrhoeae. The sepuences used are given in Table IX. The map positions for the laz region (example 25) are according to Gotschlich, E. C.; and M. E. Seiff. FEMS Microbicl. Letts. 43:
( 1987). The map positions for the P.it/opa region (examples 26 & 27) are according to Stern, A., M. Brown, P. Nickel, and T. F. Meyer. Cell 47: 61-71 ( 1986). The map positians for the pilin region.(examples 28 & 29) are according to Meyer; T. F., E. Biliyard, R. Haas, S. Storzbach, and M. So. Proc. Natl. Acad.
Sci. USA
81: 61 10-61 14 ( 1984).
~
27.1: FL-AATATTTGCG CGGCGGGGAC GG 97 27:2: GTCCCCGCLG ~GCAAATATT -FL 98 laz 27:3: TGCCCTAATA TTAAAGAAAT AGGTT-BIO 99 27:4: BIO-AACCTATTTC TTTAATATTA GGGCAG 100 66.1: FL-GCCATATTGT GTTGAAACAC CGCCC 101 66.2: 'CGG'fGTTTCA ACACAATATG GC-FL 102 opa 66.3: AACCCGATAT AATCCGCCCT T-BIO 103 66.4: ' B10-AAGGGCGGAT TATATCGGGT TCC 104 1 14.1: FL-CAACATCAGTG AAAATCTTTT TTTAACC 105 1 14.2: TTAaAAAAAG ATTTTCACTG ATGTTG-FL 106 opa 1 14.3: TCAAACCGAA TAAGGAGCCG A-BI0 107 1 14.4: 810-TTCGGCTCCT TATTCGGTTT GACC 108 i1~~3 93/7 ~ ~ P(.°1'/gJS92/(DS477 822. i : FL-TGATGCCAGC TGAGGCAAAT TAGG 109 822.2: TAATTTGCCT CAGCTGGCAT CA-FL 1 10 pi 1 i n a 822.3: TTAAATTTCA AATAAATCAA GCGGTA-BIO1 1 1 S 822.4: BI0-TACCGCTTGA TTTATTTGAA ATTTAAGG1 12 933.1: FL-CGGGCGGGGT CGTCCGTTCC 1 13 933.2: AACGGACGAC CCCGCCCG-FL 1 14 pilin 933.3: TGGAAATAAT ATATCGATI'-BIO 1 15 933.4: BIO-AATCGATATATTATTTCCAC C 1 16 a The sequences shown are a consensus of all published N, gonorrhoeae pitin genes and when taken in total do not exactly match any one particular sequence.
~x~m~rle 25.
Double gap LCR (DG2,1 ) was performed for 35 cycles consisting of a 30 '~ second incubation at 85'C and a 60 second incubation at 5~d°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. All NeisseriaDNA's were tested in the presence of 324 ng human placenta DNA. The oligonucleotides used (see Table IX, above) are specific for map positions 27-74 within the Neisseria gonorrhoeae lay gene. Reactions were run in a buffer identical to that described in Example 3 except that 5 x 101 1 molecules each of oligonucleotides 27.1, 27.2, 27.3, 27.4 (ID Nos. 97-100, respectively), and 1 ~M 2'-deoxycytidine S'-tr~phosphate w ere used.
Following amptific~tion, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
pig o y~e (Amount) Neisseria gonorrhoeae (200 cells) 576 Neisseria meningitidis (2 x 106 cells) 6 3E, Neisseria lactamica (2 x 106 cells) 5 Human placenta (320 ng) ~'~,~~ 1R
Double gap LCR (D~3,2~ f~as performed for 27 cycles consisting of a 30 40 second incubation at 85'C and a 60 second incubation at 57°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. All Neisseria DNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX; above) are specific for map positions 66-1 13 within the Neisseria gonorrhoeae opa gene. Reac ions were run in a buffer identical to that described in 1~0 93/4044? ~ ~ ~ ~. ~ ~ ~ ' , PG'r'1US92/05~~7 Example 3 except that S x 101 1 molecules each of oligonucleotides 66. t , 66.2, 66.3, 66.4 (ID Nos. 101-104, respectively), and 1~M 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactiorys were diluted t:i with IMx diluent buffer, S and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
_Dj~A ~ourr.e ~~ o m ) Neisseria ganorrhaeae 1200 cells) t ~ 10 Neisseria meningitides (2 x 106 cells) 18 Neisseria lactamica (2 x 106 cells) 8 Human placenta (320 ng> 6 E-x~OBle 2'7.
Double gap LCR (DG2,2> vvas performed for 35 cycles consisting of a 30 second incubation at 85°C and a 60 second incubation at 54°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. Ati NeisssriaDNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX, above) are specific for map positions 114-16S within the Neisseria gonorrhaeae opa gene. Reactions were run in a buffer identical to that described in Example 3 except that S x 101 1 molecules each of oligonucleotides 1 14.1, 1 14.2, t t 4.3, t 14.4 ( I D Nos. t 05-108, respectively), and 1 uM 2'-deoxyguanosine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification pr~ducts were detected via a sandwich immunoassay performed using the Abbott iMx automated immunoassay system.
ANA o ~,~~5,~~"ount) B, Neisseria gdnarrhoeae (200 cells) 306 Neisseria meningitides (2 x 106 cells) 10 Neisseria lactamica (2 x 106 cells) 10 Human placenta (320 ng) ~.Im~le-2_~- ' Double gap LCR (DG2,2> was performed for 35 cycles consisting of a 30 second incubation at 85°C-and a 6Q second incubation at 50°C.
Reactions were set up in duplicate with genomic DNA from the indicated cells. A11 Neisseria DNA's were tested in the presence of 320 ng human placenta DNA. The oligonucleotides used (see Table IX; above) are specific for map positions 822-873 within the Neisseria gonarrhoeae piiin gene. Reactions were run in a buffer identical to that described in w~ q~iooa~~ ~ ~ ~ ~ ~ ~ ~ Pc-rius9~oosa~'~
Example 3 except that 5 x i 01 1 molecules each of oligonucleatides 822.1, 822.2, 822.3, 822.4 (ID Nos: 109-1 12, respectively), and 1~M 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with IMx dtluent buffer, S and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~N ~~,~ . .fount ) 8 Neisseria gonorrhoeae (200 cells) 1291 Neisseria meningitides (2 x 106 cel)s) 27 Neisseria lactamica (2 x t 06 cells) 22 Human placenta (320 ng) 1 1 1 S ~xa y a 29.
~; ~ . Double gap LCR (DG2,2> is performed for 30 - 50 cycles as in earlier examples. The oligonucleotides used (~~~e Table iX, above) are specific for map positions 933-973 within the Neisseria gonorrhoeae pitin gene. Reactions are run in a buffer identical to that described in Example 3 except that approximately 5 x 101 1 molecules each of oligonucleotides 933.1, 933.2, 933.3, 933.4 (ID Nos. 1 1 16, respectively), and 1 uP~4 2'-deoxyguanosine 5'-triphosphate are used.
Following amplification, reactions are diluted 1:1 with IMx diluent buffer, and the LCR amplification products are detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Examples 30 ahd 31 relate to the detection of Sorrel is burgdarferi, the causative agent of "Lyme'° disease. The sequences used, their map position according to Rosa; P,A., and T:G. Schwan. J: Infect. Dis. 160:1018-1029 (1989), and their Sequence iD Nos. are: given in Table X, below. .
Table X:
~1~P.PQ S SEA EU NCE- SEG? I
D No.
S. t : FL-AAAACGAAGA TACTAAATCT GTAA1 17 5.2: ACAGATTTAG TATCTTCGTT TT-FL 1 18 5.3: CCAGAAACAC CTTTTGAATT AA-SIC!1 19 5.4: B60-TTAATTCAAA AGGTGTTTCT 120 GCAA
181.1: FL-CATCTTTTGG AGCTAAATAT AAG i 2 l 181.2: TTATATTTAG CTCCAAAAGA.TGC~FL 122 181.3: TTGGATTAAC AAAAATAAAC GAT-BIO123 181.4: BIO-TCGTTTATTT TTGTTAATCC 124 AAG
.~.,: , ;., '.;, . ...:.; - :. ;:v ~:_ . ~ . :. ::"
PCT/LJ~92/05~'~7 dVfJ 93/0047 Double gap LCR (DG2,2> was performed for 45 cycles consisting of a 25 second incubation at 8S'C and a 70 second incubation at 50'C. Reactions were set up in S duplicate with either 0'or 102 target molecules. The target DNA was a random Borrelia burgdorferi genomic sequence cloned into pUC 18. Negative reactions contained 10 nanbgrams of calf thymus DNA. Positive reactions contained 102 Borrelia target DNA molecules in 10 nanograms of calf thymus DNA. The oligonucleotides used are listed in Table X, and are specific for map positions 5-53 of the cloned Borrelia genomic DNA. Reactions were run in a buffer identical to that described in Example 1 except that 3 x 10~? molecules each otigonucleotides 5.1 and 5.3 tID Nos. 1 i7 and 1 19, respectively), 2 x 102 molecules each otigonucleotides 5.2 and 5.4 (!D Nas. 1 18 and120, respectively>, 1032 units of Thermos thermophilus DNA lipase, and 1 uM 2'deoxythymidine 5'-triphosphate were used.
. Following amplification, reactions were diluted 1:1 with IMx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
Number of target molecules 0 6.0 i 02 152.0 Double gap LCR (DG 1,1 > was performed for 45 cycles consisting of a 65 second incubation at 85°C and a 60second incubation at 50°C. Reactions were set up in duplicate with either 0 or 102 target molecules. Negative reactions contained nanograms of salmon sperm DNA. Positive reactions contained 102 8orrelis target ~NA moteeuies in t 0 nanograms of salmon sperm DNA. The oligonucleotides used are listed in Table X; and are specific for map positions i 81-228 of the cloned Borrelia genamic DNA. Reactions were run in a buffer identical to that described in Example 1 except that 1 x 10~~ molecules each oligonucleotides 18i.1, 181.2, 181.3, 181.4 E!D Nos. i21-124, respectively), 500 units of Thermos thermophilus DNA lipase, 1.6 units of Thermos DNA polymerase (MBR), and t p.M 2'-deoxycytidine 5'-triphosphate were used.
Following amplification, reactions were diluted 1:1 with !Mx diluent buffer, and the LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx automated immunoassay system.
~~ g~~o~a~ ~ ~. ~. ~. ~ ~ 2 Pcrius~~i~sa~' Number of ar~i' mot~c~.~l~~ Vie.
p S.0 319.0 The above examples serve to illustrate the 'invention, but are not intended to limit it in any way, the felt scope of the tnvention being dettned by the appended claims.
i,r' r V4~~ ~3/0~447 I'CT/~JS92/0~~77 A.
AZVSF2CG Subxequence Sites Tbusxday, June a?, 1991 8:40 AH
Sequence Range: 991 to 2299 Strt #Res.
Bos. Subseq Ioc. Hatched Subseq Found ,Search with DG 5 sites 2,2 (A): $oundm 811 ~G 2v2 (A) (part #1) 811 B/6 ATTAAG
+
816 DG 2,2 (A) .. , ~
(part #1) 816 6/6 CETAAT
~
850 DG; 2.2 (A) (part #1) 850 6/6 ~rxAAG
+
e55 ~c z,z (A) (part #1) 855 6/6 CTTAAC
~
1964 DC 2,2 (A) (part #1) 1964 6l6 GTTAAG
+
1969 35C 2,2 (A) (Past ~Il? 1969 6l6 CTTAAC
.
Search wl.th DG 14 2.2 (C)a sites found..
854 DO; 2,2 (C) (past 1~1) 854 6/6 ACGCCA
+
859 ~ 2' Ip~. #1) 059 6/6 TCGCCT
-934 DG 2,2 (C) -(part #1) 934 6/6 T:.GCCT
+
939 ~c z,z (c) (part #1) 939- 6/6 ACGCCA
-123a ~ z,2 (c) (part #1) 1230 s/s A~cccA
+
1235 d7G 2,2 (C) (pazt #1) 1235 6/6 TCGCCT
-laal ~c z,a (c) (part #1) 1471 6/6 31GGCCA
+
1496 DG 2.2 ICD _ ~ v .
(part #1) '1476 6/6 TJGCCT
-1864 DG 2,2 (C) (part #1) 1864 6/6 CGGCCA
+
1$69 DG 2v2 (C) (part #1) 1869 6/6 TCGCCC
~-2108 TsG 2.2 (C) (part #1) 2108 6/6 3'GGCCT
~+
2113 DG 2,2 (C) (part #1) 2113 6/6 AG~GCCA
-2126 DG 2,2 (C) (paict #I) 2126 6/6 AGGCCA
+
2131 DG 2,2 (C) (part #1) 2231 6/6 ~'GGCCT
~-Sea=ch vith DG 2 sites 2,2 (G), founcL
1689 DG 2,2 (G) (part #1) 1689 6/6 ACCCCT
+
1694 DG 2,2 (G) (part #1) 1694 , s/6 ACCGGT
-Search with DC 4 sites 2,2 (T): found_ 824 L7C 2,'2 (T) (past #1) 824 6l6 G~1TTA
+
829 DC 2.2 (T) (part #i) sag s/s TAaTTc -1975 DG 2,2 (T) (part #1) 1975 6/6 c..l:,TTG
+
1980 I7G 2,2 (T) (part #1) 1980 6/b CAATTG
-APPENDIX A
Claims (15)
1. A method for detecting the presence of a target nucleic acid sequence in a sample, said method employing the ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target sequence, said method comprising:
(a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula:
5'-(N)n XE p.F q Z(N)k-3' wherein E represents any base, F represents any base except E, p and q are independently integers from 1 to 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected;
(b) providing a plurality of each of four probes having the formulas:
A: 5'-(N)h.uparw. X Ep-3' A': 3'-(N')n.uparw.X'-5' B: 5'-Z (N)k.uparw.-3' B': 3'-F'q Z'(N')k.uparw.-5' wherein the symbols have the same meanings given above, except that h.uparw.
and k.uparw.
need only approximate h and k, respectively, varying by no more than 25%; E', F', X' and Z' represent the complements of E, F, X and Z, respectively, and wherein at feast one of the probes is labeled with a moiety capable of detection; and also providing deoxyribonucleotide triphosphates of E' and F, a polymerase reagent and a ligase reagent;
(c) performing the following cycle at least once:
i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present, or to reorganized probes created therefrom;
ii) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F
deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerase reagent by adding E' deoxyribonucleotide triphosphates to its 3' end;
iii) ligating extended probe A to probe B, and extended probe B' to probe A', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
(d) separating reorganized probe molecules from unreorganized labeled probes; and (e) detecting the presence of said label in the reorganized or fraction as a measure of the presence of the target sequence.
(a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula:
5'-(N)n XE p.F q Z(N)k-3' wherein E represents any base, F represents any base except E, p and q are independently integers from 1 to 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected;
(b) providing a plurality of each of four probes having the formulas:
A: 5'-(N)h.uparw. X Ep-3' A': 3'-(N')n.uparw.X'-5' B: 5'-Z (N)k.uparw.-3' B': 3'-F'q Z'(N')k.uparw.-5' wherein the symbols have the same meanings given above, except that h.uparw.
and k.uparw.
need only approximate h and k, respectively, varying by no more than 25%; E', F', X' and Z' represent the complements of E, F, X and Z, respectively, and wherein at feast one of the probes is labeled with a moiety capable of detection; and also providing deoxyribonucleotide triphosphates of E' and F, a polymerase reagent and a ligase reagent;
(c) performing the following cycle at least once:
i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present, or to reorganized probes created therefrom;
ii) using target sequence or reorganized probes created therefrom as template, extending probe A with said polymerase reagent by adding F
deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerase reagent by adding E' deoxyribonucleotide triphosphates to its 3' end;
iii) ligating extended probe A to probe B, and extended probe B' to probe A', using said ligase reagent to form reorganized probe molecules; and iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
(d) separating reorganized probe molecules from unreorganized labeled probes; and (e) detecting the presence of said label in the reorganized or fraction as a measure of the presence of the target sequence.
2. The method according to claim 1 wherein F is E', whereby step b) requires adding the deoxyribonucleotide triphosphate of only E'.
3. The method according to claim 1 or 2 wherein p and q are independently integers between 1 and 3, inclusive.
4. The method according to claim 1 wherein the 5' end of probe A and the 3' end of probe A' are labeled with a first hapten.
5. The method according to claim 4 wherein the separation step (step d) comprises capturing on a solid phase said first hapten and its associated probe.
6. The method according to claim 1 wherein the cycle of step c) is repeated from about 20 to about 60 times.
7. A diagnostic kit for the detection of a target nucleic acid sequence in a sample, said nucleic acid having a target sequence comprising:
5'-(N)h XE p.F q Z(N )k-3' wherein E and F independently represent any base, p and q are independently integers from 1 to about 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected, said kit comprising in combination:
(a) four'probes, having the formulas:
A: 5'-(N)h.uparw.XE p-3' A': 3'-(N')h.uparw.X'-5' B: 5'-Z (N)k.uparw.-3' B': 3'-F' q Z'(N')k.uparw.-5' wherein the symbols have the same meanings given above, except that h.uparw.
and k.uparw.
need only approximate h and k, respectively, varying by no more than 25%; E', F', X' and Z' represent the complements of E, F, X and Z, respectively, and wherein at least one of the probes is labeled with a moiety capable of detection;
(b) a polymerise reagent capable of extending probe A with said polymerise reagent in a target dependent manner by adding F
deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerise reagent in a target dependent manner by adding E' deoxyribonucieotide triphosphates to its 3' end, thereby to render the primary probes ligatable to one another when hybridized to target and, optionally, to render the secondary probes ligatable to one another when hybridized to target;
(c) deoxyribonucleotide triphosphates of E' and F; and (d) a ligase reagent for ligating the extended probe A to probe B, and extended probe B' to probe A', thereby to form reorganized probe molecules.
5'-(N)h XE p.F q Z(N )k-3' wherein E and F independently represent any base, p and q are independently integers from 1 to about 10, X represents any base except E or F', Z
represents any base except F or E', N represents any base, and h and k are independently integers from 5 to 100, provided the sums (p+h) and (q+k) each are greater than 10, wherein (N)h and (N)k represent sequences that are characteristic for the target sought to be detected, said kit comprising in combination:
(a) four'probes, having the formulas:
A: 5'-(N)h.uparw.XE p-3' A': 3'-(N')h.uparw.X'-5' B: 5'-Z (N)k.uparw.-3' B': 3'-F' q Z'(N')k.uparw.-5' wherein the symbols have the same meanings given above, except that h.uparw.
and k.uparw.
need only approximate h and k, respectively, varying by no more than 25%; E', F', X' and Z' represent the complements of E, F, X and Z, respectively, and wherein at least one of the probes is labeled with a moiety capable of detection;
(b) a polymerise reagent capable of extending probe A with said polymerise reagent in a target dependent manner by adding F
deoxyribonucleotide triphosphates to its 3' end, and extending probe B' with said polymerise reagent in a target dependent manner by adding E' deoxyribonucieotide triphosphates to its 3' end, thereby to render the primary probes ligatable to one another when hybridized to target and, optionally, to render the secondary probes ligatable to one another when hybridized to target;
(c) deoxyribonucleotide triphosphates of E' and F; and (d) a ligase reagent for ligating the extended probe A to probe B, and extended probe B' to probe A', thereby to form reorganized probe molecules.
8. The kit according to claim 7, and also comprising means for separating reorganized probe molecules from unreorganized labeled probes.
9. A composition of matter useful for detecting the DNA of HIV 1, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table IV and identified by sequence ID Nos: 1, 2, 3 and 4; 5, 6, 7 and 8; 9, 10, 11 and 12; 13, 14, 15 and 16; 17, 18, 19 and 20; 21, 22, 23 and 24; 25, 26, 27 and 28; 29, 30, 31 and 32; 33, 34, 35 and 36; 37, 38, 39 and 40;
41, 42, 43 and 44; and 45, 46, 47 and 48.
41, 42, 43 and 44; and 45, 46, 47 and 48.
10. A composition of matter useful for detecting the DNA of HIV 2, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table V and identified by sequence ID Nos: 49, 50, 51 and 52; 53, 54, 55 and 56; and 57, 58, 59 and 60.
11. A composition of matter useful for detecting the DNA of HPV, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table VI and identified by sequence ID Nos: 61, 62, 63 and 64;
65, 66, 67 and 68; 69, 70, 71 and 72; and 73, 74, 75 and 76.
65, 66, 67 and 68; 69, 70, 71 and 72; and 73, 74, 75 and 76.
12. A composition of matter useful for detecting the DNA of HSV, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table VII and identified by sequence ID Nos: 77, 78, 79 and 80;
and 81, 82, 83 and 84.
and 81, 82, 83 and 84.
13. A composition of matter useful for detecting the DNA of Chlamydia trachomatis, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table VIII and identified by sequence ID
Nos: 85, 86, 87 and 88; 89, 90, 91 and 92; and 93, 94, 95 and 96.
Nos: 85, 86, 87 and 88; 89, 90, 91 and 92; and 93, 94, 95 and 96.
14. A composition of matter useful for detecting the DNA of Neisseria gonorrhoeae, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table IX and identified by sequence ID
Nos: 97, 98, 99 and 100; 101, 102, 103 and 104; 105, 106, 107 and 108; 109, 110, 111 and 112; and 113, 114, 115 and 116.
Nos: 97, 98, 99 and 100; 101, 102, 103 and 104; 105, 106, 107 and 108; 109, 110, 111 and 112; and 113, 114, 115 and 116.
15. A composition of matter useful for detecting the DNA of Borrelia burgdorferi, said composition comprising a mixture of four DNA probes, said probes being selected from the probe sets listed in Table X and identified by sequence ID
Nos:
117, 118, 119 and 120; and 121, 122, 123 and 124.
Nos:
117, 118, 119 and 120; and 121, 122, 123 and 124.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/722,798 US5427930A (en) | 1990-01-26 | 1991-06-28 | Amplification of target nucleic acids using gap filling ligase chain reaction |
US722,798 | 1991-06-28 | ||
PCT/US1992/005477 WO1993000447A1 (en) | 1991-06-28 | 1992-06-26 | Amplification of target nucleic acids using gap filling ligase chain reaction |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2111962A1 CA2111962A1 (en) | 1993-01-07 |
CA2111962C true CA2111962C (en) | 2004-08-24 |
Family
ID=24903429
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002111962A Expired - Fee Related CA2111962C (en) | 1991-06-28 | 1992-06-26 | Amplification of target nucleic acids using gap filling ligase chain reaction |
Country Status (7)
Country | Link |
---|---|
US (1) | US5427930A (en) |
EP (1) | EP0596918B1 (en) |
JP (1) | JP3330599B2 (en) |
CA (1) | CA2111962C (en) |
DE (1) | DE69229999T2 (en) |
ES (1) | ES2138974T3 (en) |
WO (1) | WO1993000447A1 (en) |
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