WO1993011266A1 - Method for the detection of mutations and monoclonal lineages by analysis of rna conformation polymorphism(s) - Google Patents

Abstract

The invention is a method to detect a mutation at a predefined position within a predetermined DNA sequence from a subject. The method comprises amplifying and transcribing the predetermined DNA squence in a sample from the subject and comparing the pattern of RNA bands in a non-denaturing gel with the RNA band pattern of the same predetermined DNA sequence absent the mutation wherein any differences detected in the compared patterns indicate the presence of mutation at the predefined position in the predetermined DNA sequence. The invention also provides a method for detecting a clonespecific DNA sequence at a predetermined position in a monoclonal lymphoid population of cells from a subject, the method comprising amplifying and transcribing the DNA containing the predetermined position in a sample from the subject and comparing the RNA band pattern in a non-denaturing gel with the pattern of bands of RNA transcribed from the DNA sample known to contain the predetermined position without the clonespecific sequence wherein the detection of differences in the compared band patterns indicates the presence of the monoclonal lymphoid population of cells.

Description

METHOD FOR THE DETECTION OF

MUTATIONS AND MONOCLONAL LINEAGES

BY ANALYSIS OF RNA CONFORMATION POLYMORPHISM (S)

This invention was made in the course of work under Grant CH-1 and BC-561 from the American Cancer Society. The United States government has certain rights in this invention.

Background of the Invention

Throughout this application various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, immediately preceding the claims.

Point mutations of a single base pair are often associated with the pathogenesis of both inherited and acquired diseases. Recent well-known examples are that of the ras oncogene, which is activated by point mutations (1) and the p53 tumor suppressor gene, which in many common types of cancers has been found to contain single base mutations (2,3). For expansion of knowledge in this area, strategies for rapid and reliable detection of point mutations in specific genes would obviously be of great value. A worthwhile goal also is to find techniques that are adaptable for gene analysis in cleanicla laboratories to allow routine and inexpensive screening of individuals for the presence of specific mutations in their genes. Numerous methods are currently available for detection of point mutations, each of them having advantages as weil as drawbacks (4). DNA sequencing is the most direct method and permits unequivocal identification of the specific base substituted at the point mutation. However, sequencing is very labor intensive and is not practical either for screening a large number of samples or for searching long stretches of DNA for mutations. Sequencing as a tool for characterizing mutations is most effective when used in conjunction with a non-specific screening method that detects and fixes the approximate location of the mutations. Most such methods rely on oligonucleotide mismatch analysis. A probe of known sequence is hybridized to a region of the gene of interest. Base pair mismatches within the region covered by the probe can be detected by various chemical and physical methods. However, RNase treatment of the duplex does not cleave all mismatches (5). Denaturing gradient gel electrophoresis (6) or chemical cleavage (7) may detect a greater percentage of mismatches but these methods are more complicated and difficult to use.

Recently, Orita et al. reported that single base substitutions in short DNA segments (up to about 250 bp) can be detected as shifts in electrophoretic mobility (8, 9).

Presumably, the base substitution causes the DNA segment to assume a unique folded conformation, which alters its mobility on a non-denaturing gel compared to the corresponding unmutated DNA segment. The strategy is to amplify the desired segment of a gene by the PCR (10), and then to compare the migration pattern of the denatured DNA with that of a reference segment of known sequence. The "single strand conformational polymorphism" (SSCP) assay is simple, rapid, and sensitive, and has now been used for detection of point mutations in several studies (11, 12, 13, 14, 15). However, because this is still a relatively new method, it is uncertain what percentage of mutations will be. detected by DNA SSCP analysis (12). Cawthon et al. (15) noted that in some cases, one strand of the mutated DNA has an altered electrophoretic mobility but the other strand, even though it has the complementary mutation, continues to migrate with the same mobility as the normal allele. With dehydrofolate reductase (DHFR) gene mutants, it was found that in some instances DNA SSCP analysis did not detect point mutations even in short DNA segments.

Traditionally, clonal lymphoproliferative disorders are diagnosed by morphology, immunogistochemistry and fluorescence activated cell sorting. On the molecular level, monoclonal populations can be detected by Southern blot analysis of T-Cell receptor (TCR) or immunoglobulin gene rearrangements. However, Southern blot analysis is associtaed with severe limitations and is not feasible in many clinical situations. Relatively large amounts (several micrograms) of well preserved DNA are required and it is often difficult to obtain, e.g., from paraffin-embedded material). In addition, this assay is labor and time intensive.

Summary of the Invention

A method to detect within a predetermined DNA sequence from a subject, the presence at a predefined position, a mutation, is provided by this invention. This mehtod comprises obtaining from the subject a sample containing the predefined DNA sequence, amplifying this DNA sequence, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands obtained with the pattern of bands obtained by separating on a non-denaturing gel RNA transcribed from a sample known to contain the predetermined DNA sequence without the mutation at the predefined position, and detecting any differences in theses patterns, the presence of a difference in the patterns indicating the presence of a mutation at the predefined position of the predetermined DNA.

This invention also provides a method for detecting in a monoclonal lymphoid population of cells from a subject, the presence at a predetermined position of a clonespecific DNA sequence, which comprises obtaining from the subject a DNA sample containing the predetermined position, amplifying this DNA, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands obtained by separation on non- denatureing gel to RNA transcribed from a DNA sample known to contain the predetermined position without the clonespecific sequence and detecting any differences in the patterns, the presence of a difference in the patterns indicating the presence of the monoclonal lymphoid population of cells. Brief Description of the Figures

Figure 1. Comparison between DNA SSCP and RNA SSCP analysis of p53 gene point mutations.

Wild-type p53 plasmid, p53-SN3 (lanes 2, 4, 6) and a synthetic 95 mer mutant p53 template (codon 175, CGC→CAC, Arg→His) (lanes 1, 3, 5) were PCR-amplified using the primer pair p53-75 and p53-76 (see Materials and Methods - Experiment No. 1). The predicted DNA fragment would be 95 bp spanning positions +1036 to +1130 plus the T7 promoter sequence for a total of 118 bp. For DNA SSCP (lanes 1-4) the ³²P-end-labeled primers were used for PCR and for RNA SSCP (lanes 5 and 6), PCR amplified fragments were transcribed into a 101 nucleotide (95 nucleotide coding sequence plus 6 nucleotide transcription start sequence) fragment of uniformly ³²p-labeled RNA. Non-denatured (lanes 1 and 2) and denatured PCR-amplified DNA products were applied to a native 8% polyacrylamide gel containing 10% glycerol and electrophoresed at room temperature (20-22°C) at 25 W constant power according to the conditions of Orita et al. (8). RNA (lanes 5 and 6) was applied to the same gel without denaturation.

Figure 2. Comparison of DNA SSCP and RNA SSCP analysis of a DHFR gene mutation. The cDNA libraries from HCT-8 cells (lanes 1, 4, 7) and from HCT-8R4 cells (lanes 2, 5, 8) containing a DHFR gene with a T→C mutation at position 91 (codon 31, TTC→TCC, Phe→Ser), and plasmid pHD84 (lanes 3, 6, 9) containing the same mutation as the HCT-8R4 cells were PCR-amplified using primer pair DHFR-70 and DHFR-72. The anticipated fragment would have 163 bp of coding sequence spanning positions +38 to +200, plus 20 bases from the T7 promoter sequence for a total of 183 bp. The labeling of each fragment was the same as in Figure 1. Denatured (lanes 1-3) and non-denatured (lanes 4-6) PCR-amplified DNA products were applied to a native 6% polyacrylamide gel containing 10% glycerol, and electrophoresed at room temperature (21-31°C) at 20 W constant power. Transcribed RNAs of 166 nucleotides (coding sequence plus 3 bases from the transcription start site) (lanes 7-9) were applied to the same gel without denaturation. Direct sequence analysis of the two lower bands of the RNA of HCT-8 cells (lane 7) showed that the lower band had a wild-type sequence while the upper band has a silent A→G transition at codon 32(AGA→AGG, Arg→Arg).

Figure 3. Demonstration of conformational equilibration of RNA. The same wild-type and mutant p53 templates as shown in Figure 1 where PCR-amplified and transcribed into RNA. Uniformly ³²P-labeled RNAs were applied to a native 8% polyacrylamide gel without glycerol and electrophoresed at 5-7°C at 25 W constant power (A). The bands numbered 2, 3, and 4 from wild-type RNA and band 1 (small arrow in A) from mutant RNA were excised and each RNA was extracted from the gel. The extracted RNA was applied to gels B and C with not further treatment (lanes a, d, g, j) or after heating at 90ºC for 3 minutes (lanes b, e, h, k) or treatment with 70% formamide (lanes c, f, i, l). Electrophoresis conditions for B and C were the same as for A. Figure 4. RNA-SSCP analysis of point mutations in mouse genomic DHFR (A), mouse thymidine kinase (B), and human p53 (C).

A. Genomic DNA containing the wild-type DHFR gene from mouse L1210 cells (lanes 1 and 4) and genomic DNA from two individual cell lines containing identically mutated DHFR genes (23) (codon 15, GGG→TGG, Gly→Trp) (lanes 2 and 3) were PCR-amplified using the primer pair DHFR-85 and DHFR-86 and then transcribed to RNA. These primers are expected to generate a fragment of 177 nucleotides spanning a region of the genomic DNA from positions -77 to +100 plus a 6 base transcription start sequence for a total of 183 nucleotides.

B. The cDNA library from mouse mammary adenocarcinoma FM3A wild-type (lanes 1 and 3) and from FM3A (TK-) cells (lane 2) containing a mutated thymidine kinase gene (codon 158, G→C, Arg→Pro) were PCR-amplified using primer pair TK-5 and TK-43 and then transcribed to RNA. The anticipated fragment would be 257 nucleotides, corresponding to positions +319 to +575 of the thymidine kinase cDNA plus a 6 base transcription start sequence for a total of 263 nucleotides.

C. The wild-type p53 plasmid pC53-SN3 (lane 2) and mutant p53 plasmid p53-SCX3 (codon 143, C→T, Val→Ala)

(25) (lanes 1 and 3) were PCR-amplified using primer pair p53-77 and p53-79 and then transcribed into RNA.

The anticipated fragment of 152 nucleotides would correspond to positions +912 to +1063 plus a 6 base transcription start sequence for a total of 158 nucleotides. The RNAs were applied to a native 8% polyacrylamide gel without glycerol and electrophoreses at 5-7ºC at 25 W constant power. Figure 5. RNA SSCP analysis of several mutations in the human DHFR gene.

The plasmids were PCR-amplified using primer pair DHFR-70 and DHFR-71. The PCR-amplified DNAs were transcribed into uniformly-labeled ³²P-RNA. The anticipated fragment of 134 nucleotides would span positions +38 to +171 of the DHFR cDNA plus a 3 base transcription start sequence for a total of 137 nucleotides. The RNAs were applied to a native 8% polyacrylamide gel without glycerol and then electophorased at 6-7°C at 25 W constant power. Lane wt (wild-type): wild-type DHFR plasmid pHD84. Lane 1: Phe 22. Lane 2: Met 22. Lane 3: Trp 22. Lane 4: double mutant Phe 22/Ser 31. Lane 5: Ser 31. Lane 6: Ser 34. For a more complete description of the mutant DHFR plasmids, see Materials and Methods - Experiment No. 1.

Figure 6. Non-radioactive visualization of RNA SSCP analysis gels. Wild-type p53 plasmid p53-SN3 (lane 1) and synthetic mutant p53 templates p53-80 (lane 2) were PCR-amplified and transcribed in Figure 1. Wild-type DHFR plasmid pHD84 (lane

3) and mutant DHFR plasmid Ser 31 were PCR-amplified and transcribed as described in Figure 5. Transcribed RNAs were applied to a native 8% polyacrylamide gel without glycerol and electrophoresed at 6-7ºC at 25 W constant power. The gel was then stained with ethidium bromide and the photograph was taken of the gel on top of a UV- transilluminator.

Figure 7. TCR-gamma junctional sequences were amplified by PCR, the reaction product was transcribed into complementary RNA. The different cRNA conformational polymorphisms were analyzed by electrophoresis on a 8% non-denaturing polyacrylamide gel electrophoresis. Lane 1: Marker substance. Lane 2, 3: Analysis of polyclonal lymphoid populations of 2 healthy individuals. Lane 4, 5, 6: three patients with acute leukemia. Lane 7: patient with mycosis fungoides, DNA isolated from paraffin embedded, formalin fixed tissue. The rearrangement of the TCR genes was proven by cloning and direct sequence analyses prior done to this experiment. Figure 8. The junctional sequences of IgH gene rearrangements were amplified by PCR and transcribed into complementary RNA. The reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel. Lane 1, 2, 3: patients with acute lymphocytic leukemia. Lane 4: Marker substance.

Detailed Description of the Invention

This invention provides a method to detect within a predetermined DNA sequence from a subject, the presence at a predefined position, a mutation, which comprises obtaining from the subject a sample containing the predefined DNA sequence, amplifying this DNA sequence to obtain multiple copies of the sequence, transcribing the amplified DNA to RNA, separating this RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined or "characteristic" pattern, comparing this pattern with the pattern of; RNA obtained by separating on a non-denaturing gel RNA transcribed from a sample known to contain the predefined DNA sequence without the mutation at the predetermined position, and detecting any differences in these two patterns, the presence of a difference in the patterns indicating the presence of a mutation at the predetermined position of the predefined DNA. As used herein, the term "mutation" means any change in the sequence of DNA. For the purposes of this invention, this DNA is genomic DNA, single-stranded DNA, double-stranded DNA, plasmid DNA, bacterial DNA, parasitic DNA or viral DNA.

The method of the subject application is specifically powerful for the detection "point mutations". A point mutation is an alteration that changes a single base pair in the DNA sequence. Historically, such single or point mutations have been difficult to detect.

For the purposes of this invention, a suitable DNA sample is any sample containing the DNA sought to be assayed. As used herein, "amplified" means multiply the copy number of the sequence either by enzymatic amplification (polymerase chain reaction or "PCR") or by traditional cloning techniques. When the DNA is amplified by traditional cloning techniques, the predetermined sequence is isolated, utilizing restriction enzymes that cut the DNA at predetermined, specific locations to create restriction enzyme fragments. These fragments are isolated by separation techniques well known to those of skill in the art, such as separating the restriction enzyme fragments by running them on an agarose gel. The fragments are purified from the gel, by techniques well known to those of skill in the art, and the fragment containing the predefined position is inserted into a recombinant cloning vector such as a virus or plasmid. Suitable host cells are then transformed with these cloning vectors. The host cells are then grown under suitable conditions such that the DNA fragment is multiplied. The vector DNA is then isolated from the host cell and the inserted DNA is then removed from the vector by the use of restriction endonucleases, run out on an agarose gel and subsequently purified from the gel.

However, in the preferred embodiment of this invention, the DNA is amplified utilizing polymerase chain reaction ("PCR"). When using PCR, a suitable primer is selected which neighbors the predefined position. This primer may include a T7 polymerase promoter sequence on one end of the primer. The DNA is then amplified and transcribed to RNA using methods well known to those of skill in the art. In the most preferred embodiment of this invention, the primer contains not only the T7 polymerase sequence but also a "clamping sequence" which increases the yield of the RNA product and is transcribed along with it.

However, when the DNA is not amplified by PCR, it also is necessary to transcribe the predetermined DNA containing the predefined position to RNA by the use of RNA polymerase. Methods of transcribing a DNA sample to RNA using RNA polymerase enzymes are well known to those of skill in the art.

The resulting RNA, obtained either through traditional cloning techniques or PCR, is then run on a non-denaturing gel, such as a polyacrylamide gel (PAGE), to obtain an RNA pattern characteristic of the DNA sample. Prior to separating the RNA on the gel, it may be labelled with a detectable marker, such as a radioisotope or a fluorescent label. Alternatively, if the RNA is not labeled, the resulting RNA gel may be dyed with ethidium bromide so that the RNA pattern may be visualized. This pattern is then compared to the pattern of RNA obtained from "wild-type" or "normal" DNA, i.e., the predetermined DNA sequence without the mutation at the predefined position. If a mutation is present in the DNA sample, the RNA pattern resulting from the DNA sample will be different from the wild-type. For the purposes of this invention, the subject is an animal or a mammal. In the preferred embodiment of this invention, the subject is a human patient.

This method is particularly useful for the detection of point mutations in small samples of DNA, for example, DNA obtained from paraffin-embedded material. Inherited or acquired diseases may be detected, at the genetic level, utilizing the subject invention. For example, activation of the ras oncogene, which is activated by point mutations, may be detected utilizing the subject invention.

Also provided by this invention is a method for detecting a monoclonal lymphoid population of cells, wherein the monoclonal population is the result of the presence of variable idiotypic DNA sequences between lymphoid populations. This also is a method for detecting in a monoclonal lymphoid population of cells from a subject, the presence of a predetermined position of a clonespecific DNA sequence.

This method comprises obtaining from the subject a DNA sample from a lymphoid cell, containing the predetermined position of a clonespecific DNA sequence, amplifying this DNA sequence, transcribing the amplified DNA to RNA, separating the resulting RNA on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern, comparing the pattern of bands, obtained by separation on non-denaturing gel to RNA transcribed from a DNA sample known to contain the predetermined position without the clonsespecific (i.e., DNA obtained from a polyclonal lymphoid population), and detecting any differences in the RNA patterns, the presence of a difference indicating that the sample of DNA contains monoclonal lymphoid population of cells. The band pattern on the gel is clonotypic for each individual lymphocytic clone. With this method it is possible to distinguish different clones from the same precursor.

In one embodiment of this invention, the monoclonal lymphoid population of cells are T-cells and in another embodiment of this invention the lymphoid population of cells are B cells.

When the lymphoid population of cells are T-cells, the predetermined position is the DNA sequence corresponding to the T-cell receptor gamma gene or it may be, but is not limited to, the DNA sequence corresponding to the T-cell receptor gamma junctional gene. In preferred embodiment of this invention, the variation giving rise to the clonespecific population of cells of the predetermined sequence is in the DNA corresponding to the T-cell receptor gamma variable region. For the purposes of this invention, "clonespecific" means the presence of variable sequences generated during the process of rearrangement of genes, creating unique sequences for individual lymphoid clones.

When the lymphoid population of cells are B cells, the predetermined position is the DNA sequence corresponding to the variable DNA sequences in the junctional region of immunoglobulin gene. In one embodiment of this invention, the DNA sequence correspond to CDR I, CDR II, CDR III or IgH. "Amplified" has been described hereinabove.

In another embodiment of this invention, this method further comprises labeling the RNA obtained after amplification and subsequent to transcription. The RNA may be labeled with a radioisotope or fluorescent label by methods known to those of skill in the art. Suitable lymphoid cell samples for the practices in this invention include, but are not limited to, lymphocytic paraffin-embedded material, lymph node specimens, biopsies and peripheral blood lymphocytes. When the RNA is not labelled, the gel may be stained with ethidium bromide so that characteristic bands or patterns may be identified.

When the DNA sequences are amplified by PCR, primers are created which are complementary to conserved sequences of these genes, flanking the predetermined position in the DNA sequence. In the preferred embodiment, the primers have joined to the 5' end, DNA sequences corresponding to a T7 polymerase promoter. This enables the transcription of the PCR amplified junctional sequences into RNA. However, as noted hereinabove, when the DNA is amplified by traditional cloning techniques, the amplified DNA must be transcribed to RNA by the use of RNA polymerases, using methods known to those of skill in the art. Tables I and II, below, schematically describe this invention.

When the monoclonal population in the sample consists of B cells, variable DNA sequences in the junctional regions of immunoglobulin genes (e.g., IgH, CDR I, CDR II, CDR III) may be detected by this method of conformational polymorphisms of complementary RNA of variable gene segments.

By the use of the above method, disorders such as lymphomas of B cell and T-cell lineages as well as acute lymphocytic leukemias of both lineages may be detected in various clinical specimens (biopsies, paraffin embedded, formalin fixed tissue, aspirates, and exfoliating cells).

The practice of this invention is particularly useful for the detection of monoclonal populations of B cell and T-cell lineages. It enables one of skill in the art, to compare different biopsies or samples of the same patient, obtained from different localisations or taken at different timepoint of the disease. By this method it is particularly easy to detect subclone or new clone formation in acute lymphocytic leukemias at various timepoints of disease. In cases of relapse after therapy of a lymphoma or an acute lymphocytic leukemia it is possible to compare the relapse pattern with the initial pattern. If a new clone is causing the relapse, there might be still a response to a therapeutical regiment which has been used primarily to eradicate the primary clone. If the same clone is still present, a resistance to the initial therapy is suggestive.

Further provided by this invention is a method to detect a mutation or variation in a sample of DNA, wherein the DNA sample is characterized by the presence of conserved and variable DNA sequences. This method is as the method described above for the detection of mutations. However, in this embodiment, the mutation is present in the variable sequence. In the preferred embodiment of this invention, the DNA sequence is amplified and transcribed by PCR, and the primer is complementary to the conserved sequence flanking the variable DNA sequence.

This method is useful to distinguish different viral, bacterial or parasital strains from each other, especially when such strains cannot be distinguished easily be conventional methods. The information derived from this method is useful for epidemiological studies, for diagnostics, in the treatment of infectious diseases, as well as for the development of vaccines.

The use of RNA instead of DNA was explored to determine if it would increase the sensitivity and resolution of SSCP analysis. Conversion of the PCR-amplified DNA to complementary RNA is easily accomplished by including a T7 polymerase promoter sequence on one of the PCR primers (17) and would generate a single strand, thus making denaturation of double-stranded DNA unnecessary. However, more important considerations for an RNA-based SSCP method are that 1) RNA can assume elaborate secondary and tertiary structures (18); 2) RNAs can simultaneously exist in a number of different conformations which do not equilibrate among each other unless denaturing or high temperature conditions are applied (19, 20); and 3) that the conformations even of large RNA molecules appear to be sensitive to single-base substitutions. (21). Thus, the probability of observing electrophoretic differences due to single base substitutions might be greater using RNA instead of DNA. In order to investigate the use of RNA in SSCP analysis, electrophoretic patterns of RNA molecules corresponding to normal and single-base mutated segments of the genes of DHFR, p53, and thymidine kinase were compared. It was found that the RNA generated from amplified DNA usually had numerous conformational forms that were observable by native gel electrophoresis and that single base mutations changed the distribution and the number of these forms. RNA SSCP analysis was able to detect single base substitutions in all of the segments tested including samples in which the DNA SSCP method failed to detect any change.

Experimen t Number 1 - Detection of Point Mutations Materials and Methods

Cell lines. Human colon carcinoma cell line HCT-8 was obtained from the American Type Culture Collection. The HCT-8R4 cell line was developed from HCT-8 cells by stepwise adaptation to methotrexate (MTX) (22). MTX-resistant mouse L1210 cells containing a G→T(Gly→Trp) transition at nucleotide 46 of the DHFR gene were developed in vivo (23). The mouse adenocarcinoma ceil line FM3A (TK~) was developed from wild-type FM3A cells by stepwise treatment with 5- fluorodeoxyuridine (24).

PCR templates. The p53 plasmids pC53-SN3 (wild-type) and pC53-SCX3 (Val→Ala mutant at codon 143) (25) were obtained from Dr. B. Vogeistein. Plasmid pHD84 containing the wild- type human DHFR sequence (26) was: obtained from Dr. G. Attardi. The DHFR mutants Phe 22 (Leu 22→Phe, CTG→TTT) Met 22 (Leu 22→Met, CTG--ATG), Trp 22 (Leu 22→Trp, CTG→TGG), Phe 22/Ser 31 (Leu 22-Phe, CTG-TTT; Phe 31→Ser, TTC→TCC), Ser 31 (Phe 31→Ser, TTC→TCC), and Ser 34 (Phe 34→Ser, TTC→Tcc) were synthesized by site-directed mutaginesis of wild-type DHFR cDNA contained in expression vector pKT7HDR (27) and furnished to us by Dr. Adam Dicker, Memorial Sloan-Kettering Cancer Center. Template p53-80 containing the p53 gene sequence from positions +1036 to +1130 with a G→A substitution at nucleotide 1084 (codon 175) was synthesized on an Applied Biosystems model 391 PCR-MATE DNA synthesizer by the phosphoramidite method. The sequence of p53-80 was:

CC ATG GCC ATC TAC AAG CAG TCA CAG CAC ATG ACG GAG GTT GTG AGG CA*C TGC CCC CAC CAT GAG CGC TGC TCA GAT AGC GAT GGT CTG GCC CCT. The asterisk indicates the site of the change from the wild- type sequence (28).

DNA and RNA isolation. DNA was isolated according to the procedure of Blin and Stafford (29). RNA was isolated by the AGPC method of Chomczynski and Sacchi (30).

PCR. The primers listed below were synthesized for use in the PCR. Each 5' primer had a T7 polymerase promoter sequence on its 5' end, indicated by the prefix T7. The segments in quotation marks are not part of the target gene sequence, but represent a "clamping sequence" which increases the yield of the RNA product and is transcribed along with it.

(T7 - TAA TAC GAC TCA CTA TA) p53-75: T7-"GGGAGA" CC ATG GCC ATC TAC AAG CAG TCA

p53-76: AGG GGC CAG ACC ATC GCT AT

DHFR-70: T7-"GGG" AGA ACA TGGG CAT CGG CAA GAA CG

DHFR-72: GG TCG ATT CTT CTC AGG AAT GG

DHR-71: GGT CTT CTT ACC CAT AAT CAC CAG

DHFR-85: T7-"GGGAGA" TCA GGG CTG CGA TGT CGC GCC AAA

DHFR-86: AGC CCG GCC AAT ACC TGA GCG GA

TK-5: T7-"GGGAGA" TC GAT GAG GGG CAG TTT TTT CC

TK-43: ATA CTT GTC GGC TCC GCC AAT CA

p53-77: T7-"GGG" ACA GCC AAG TCT GTG ACTTG

p53-79: PG CTG TGA CTG CTT GTA GAT GG

The PCR reactions contained 12.5 pmol of each of the primers, 2.5 ul of 10X TAQ buffer (500 mM KCI, 100 mM Tris- HCl, pH 8.3, and 0.01% gelatin), 200 uM deoxyribonucleotide triphosphates, 1.87 mM MgCl₂, 10-100 ng of template DNA and 0.63 units of TAQ polymerase (Cetus) in a total volume of 25 ul. The reaction mixture was overlaid with mineral oil. Before the TAQ polymerase was added, the mixture was heated to 95ºC for 5 minutes. The PCR conditions were 30 cycles of 1 minute at 93.5°C, 1 minute at 55°C, and 1 minute at 72°C in an Ericomp Twinblock Temperature Cycler.

When DNA SSCP was performed, both PCR primers were 5' end- labeled according to the forward reaction conditions in the BRL T4 polynucleotide kinase kit. The primers (75 pmol) were added to 30 pmol of [γ^-32P]ATP (5000 C_i/mmol) (Amersham), 5.6 ul of BRL 5X forward kinase reaction buffer (300 mM Tris-HCI, pH 7.8, 75 mM 2-mercaptoethanol, 50 mM MgCl₂ and 1.65 uM ATP) and 10 unites of T4 polynucleotide kinase (BRL) in a total volumn of 27.4 ul. The reaction was incubated for 30 minutes at 37°C and then heated for 5 minutes at 65ºC. The primers were precipitated with ethanol before use in the PCR reaction. When RNA SSCP was performed, the PCR primers were not labeled, but the PCR reaction was transcribed with T7 polymerase. To a 0.5 ml tube were added 2.5 ul of 10X transcription buffer (400 mM Tris hydrochloride, pH 7.5, 120 mM magnesium chloride, 100 mM dithiothreitol, and 10 mM spermidine), 2.5 ul of an aqueous 10 mM solution of ribonucleotides (ATP, CTP, GTP, and UTP), 0.25 ul of RNA guard (Pharmacia), 15.8 ul of diethylpyrocarbnate-treated water, 0.25 ul of [α^-32P]CTP (3000 Ci/mmol) (Amersham), 3 ul of the PCR reaction and 0.68 ul of T7 RNA polymerase (69 U/ul) (Pharmacia). The mixture was incubated for 1 hour at 37ºC. To stop the reaction, 0.75 ul of 0.5 M EDTA were added.

Gel electrophoresis. To analyze the PCR products, either end-labeled amplified DNA or uniformly-labeled transcribed

RNA were electrophoresed on a 6 or 8% polyacrylamide gel with or without 10% glycerol in a Hoeffer SE-600 dual-cooled PAGE unit. The gel dimensions were 140 x 135 x 1.5 mm. The running buffer was 89 mM Tris-borate, pH 8.3, and 2 mM EDTA. The gels were run at 25 watts constant power per plate at either 20-22°C or 6-7°C by circulating tap or ice water, respectively, through the cooling tubes of the gel apparatus. The gel running buffer was pre-equilibrated at the correct temperature for at least 1 hour. DNA SSCP analysis was performed according to Orita et al. (16). For RNA SSCP analysis, 4 ul of the T7 transcription reaction were mixed with 1 ul of 50% glycerol containing 0.25% bromophenol blue dye and electrophoresed as described above. The gel was then dried and exposed to Kodak XAR-5 film or stained wet with ethidium bromide and photographed with Polaroid 665 film.

RNA sequencing. RNA fragments were extracted from the gels by exising the portions of the gel containing the desired bands and shaking the gel pieces overnight in 0.5 M ammonium acetate. The RNA was precipitated with ethanol and then sequenced with AMV reverse transcriptase according to Stoflet et al. (17). Experimental Results

Comparison of DNA and RNA SSCP analysis. To compare RNA and DNA SSCP patterns, a 118 bp wild-type and mutant fragment of the p53 gene using the PCR was amplified. The fragments corresponded to positions +1036 to +1130 of the p53 coding region and the mutant DNA had a G to A substitution at codon 175. A portion of the double-stranded DNA was transcribed to RNA using T7 polymerase. Electrophoresis was performed using the optimal conditions suggested by Orita et al. (8). There was no discernible difference in migration between the separated wild-type and the mutant DNA strands (lanes 3 and 4 of Figure 1). On the other hand, the RNA patterns (lanes 5 and 6) were quite different. Although the major RNA bands migrated identically, the wild-type RNA displayed at least four more bands than did the RNA generated from the mutant DNA.

Wild-type and mutated DHFR gene segments were further compared by RNA and DNA SSCP analysis. The following was used: 1) a 183 bp DHFR segment spanning positions +38 to +200 of cDNA prepared from HCT-8R4 cells, which contains a known mutation at position 91 of the coding region (resulting in a substitution of serine for phenylalanine at codon 31) (22); 2) the same fragment amplified from cDNA of HCT-8 cells, which presumably had the wild-type sequence; and 3) the same segment from a vector containing the DHFR gene carrying the same mutation as the HCT-8R4 cells (27) as a control. The PCR amplified double-stranded DNA was remarkably homogenous and showed no visible secondary bands (lanes 4-6 of Figure 2). The denatured DNA did not display any noticeable difference in migration between the wild-type (lane 1) and the mutants (lanes 2 and 3). Only two bands were seen instead of the expected three bands, probably because one of the single-stranded DNA molecules migrated identically with the double-stranded DNA (the lower band). In contrast, the RNA SSCP showed a distinct difference between migration of the HCT-8 RNA (lane 7) and the two mutant RNAs (lanes 8 and 9). It was originally thought that the HCT-8 cells would contain only the wild-type sequence, and thus that the two bands visible in lane 7 were different conformations of wild-type RNA. In an effort to show that the bands shared the identical wild-type sequence, but unexpectedly, the upper band revealed a silent A to G mutation at codon 37. Thus, these two bands actually originate from alleles of DHFR found in the HCT-8 cells, but ones which give rise to the same amino acid in the DHFR protein. With respect to the present study, this result illustrates that RNA SSCP analysis was able to resolve two alleles which were not resolved by DNA SSCP.

Interconversion of RNA conformations. One of the reasons for using RNA SSCP was that numerous stable RNA conformations should exist, thus potentially adding an extra dimension of resolution to the analytical method. In order to determine whether the multiple RNA bands in Lanes 5 and 6 of Figure 1 were actually conformational isomers rather than spurious non-specific amplification bands, these bands were isolated from the gel and re-electrophoresed after various treatments. Figure 3A shows the RNA SSCP patterns of the wild-type and mutant p53 segments with the wild-type RNA run in two lanes to show that the pattern of bands was reproducible. Figure 3B shows the re-electrophoresis of some of the bands designated by numbers in Figure 3A. When band 2 (which actually consists of two closely spaced bands) was re-electrophoresed without any heating, small amounts of bands 5 and 6 were visible (lane a). Heating the material (lane b) caused more formation of products 5 and 6 as did treatment with formamide, a mild denaturing agent (lane c). Interestingly, band 2 did not give rise to any appreciable amounts of band 3 or 4. Band 3 when re-electrophoresed (lanes d-f) also gave rise mostly to band 5 and a small amount of both 4 and 6 but none of band 2. Heating also increased the conversion of band 3 to band 5. Band 4 apparently was stable conformation, because only a small amount of conversion to band 5 was observed under conditions where the other conformations underwent substantial re- equilibration. The upper band of the mutant RNA (shown by the small arrow in Figure 3A) was also isolated and was found to interconvert to bands 5 and 6, more so under mild heating (Figure 3C, lane k) or denaturing conditions (lane 1). These results demonstrate that the bands seen in the RNA SSCP gels consist of RNA conformational isomers. More examples of RNA SSCP analysis. A 177 bp fragment of the DHFR gene was amplified by the PCR suing as templates genomic DNA from wild-type L1210 cells and two MTX-resistant cell lines known to contain the same mutation in codon 15 (GGG to TGG, resulting in a Gly to Trp substitution in the protein) (230. Figure 4A shows that the pattern of the major bands of the wild-type RNA (lanes 1 and 4) differs from that of the two resistant cell lines (lanes 2 and 3). There is also a pattern of slower-migrating minor bands (not very visible in the photograph) which vary between the cell lines.

The effect of a single base mutation on RNA SSCP was examined in the mouse thymidine kinase gene. A 257-bp region of the gene covering positions +319 to +575 of the coding region was amplified. Figure 4B shows a clear difference in migration between the fragment from the wild- type cells (lanes 1 and 3) the mutant fragment containing a G to C substitution at position 505 (Arg→Pro at codon 158) (lane 2).

Figure 4C analyzes a mutation in the p53 gene consisting of a C to T substitution at codon 143 (Val→Ala). In this case, the major band of the two fragments migrated identically but the wild-type (lane 2) showed several additional minor conformations. This difference is more subtle than seen in the other examples, but nevertheless diagnostic of a different sequence.

A number of different DHFR mutants were compared in Figure 5. These mutants had been generated by site-directed mutagenesis of DHFR cDNA cloned into an expression vector. The sites of mutation were close enough so that the same set of PCR primers could be used to generate fragments containing the mutation. Each of the segments containing a single-base substitution gave a unique pattern of RNA conformations.

Visualisation of the RNA bands. One of the favorable consequences of converting PCR-amplified DNA into RNA is that a further 500-fold amplification of the PCR amplified oligonucleotide occurs in one step (17). An advantage of this amplification for SSCP analysis is that the large amount of oligonucleotide thus generated permits one to load enough RNA onto the gel so that the bands can be conveniently visualized by ethidium bromide staining. The ethidium bromide stained gels are shown in Figure 6 and correspond to lanes 5 and 6 of Figure 1 and lanes wt (wild- type) and 5 of Figure 5. Except for very faint bands, the same pattern that is seen by autoradiography is also visible by ethidium bromide staining. The RNA bands can also be visualized by UV shadowing, which provides a very rapid means of monitoring the results of the experiment. The wet gel is placed on top of a commercial silica gel plate containing fluorescent indicator, and the RNA bands become visible when placed under a hand-held UV light. Experimental Discussion

The DNA SSCP method for detecting point mutations as it is currently used depends on a single base substitution in the DNA strand causing the formation of a novel conformation sufficiently different from that of the wild-type to cause an observable change in electrophoretic migration (8, 9). Thus, a small change in the conformation of the DNA may not be detected by one-dimensional gel electrophoresis and the analysis would therefore give a false negative answer as to the presence of a mutation. However, the use of RNA in SSCP analysis, on the basis of evidence disclosed herein, shows that RNA may have multiple stable conformational states available to it (19, 20) and therefore, the probability of seeing a change in one or more of these states due to a point mutation would be greater than for the single conformation that is normally seen in DNA SSCP analysis. It was observed, for example, that human 7SL RNA is separated into 4 different conformational states by gel electrophoresis and that mutation of the RNA by deletion of small segments from certain regions eliminated 3 of the 4 conformations (19). In this study, it was shown that gel electrophoresis of RNA strands under non-denaturing conditions produced complex patterns of different conformations and that changes in these patterns were observed each time single-base substitutions were made in the RNA. The mutations caused shifts in the mobility of major conformational states, loss of some conformations, and creation of additional new conformations as well. The RNA SSCP analysis was able to detect single-base substitutions in several cases where no change in migration of the analogous mutated DNA was observed.

It is not yet clear if the RNA intrinsically has much more conformational polymorphism than DNA or whether the denaturing conditions used to separate the double-stranded DNA into single stands promotes equilibration of the molecule to a single most stable conformation. The RNA generated by T7 polymerase transcription was not subjected to denaturation before gel electrophoresis so it is possible that single-stranded DNA generated by asymmetric PCR would also display many conformational states. It is shown, however, that the different conformations of RNA can be interconverted but it is interesting to note that when each separate conformation was isolated and subjected to either heat or the denaturant, the same ratio of conformations was not obtained that was found in the transcription mixture nor did the different conformations interconvert to the same ratios of the other conformations. Thus, equilibration to ratios representative of the relative thermodynamic stabilities appears to be quite slow, probably indicative of high activation energies for the conformational transitions.

The size limit of the fragments in which DNA SSCP analysis can detect single-base substitutions is about 250-330 bp (8, 9). In this study, we did not attempt to find an upper limit to the size of the RNA fragments in RNA SSCP, but it may be similar to that of DNA SSCP. The mutation in the 250 nucleotide thymidine kinase fragment was readily observed, but a 350 nucleotide fragment of wild-type and mutant DHFR did not show any difference. However, it can be possible to analyze larger fragments by changing the PAGE conditions.

The RNA-SSCP method has several important practical advantages. First, the RNA molecules are always single- stranded and non-complementary to each other. Thus, it is not necessary to dilute the RNA before gel electrophorese to prevent reannealing as frequently occurs with denatured double-stranded DNA molecules. Much more material can therefore be loaded onto the gels, permitting the use of non-radioactive visualization methods such as ethidium bromide staining. Some sensitivity may be lost by non- radioactive visualization, but in most cases it should be sufficient to detect differences between electrophoretic patterns. Secondly, the transcription reaction to convert the PCR-amplified DNA to RNA gives uniformly radiolabeled RNA of high specific activity and thus permits the PCR reaction to be performed without radioactivity. Since the transcription is performed on a small aliquot of the PCR reaction mixture, the remaining PCR product is available for immediate sequence analysis if it appears that a mutation is present in the target segment. Thirdly, perhaps because RNA conformations are generally more stable than those of DNA, the RNA SSCP method was not as critically dependent on gel electrophoretic conditions gave the best results (7-8% PAGE, 4°C, no glycerol, run at a constant 25 watts per gel) the differences between wild-type and mutant RNA could still be observed under practically all conditions that were tried.

In summary, RNA SSCP is as simple and rapid as DNA SSCP and may detect point mutations more reliably.

Experiment Number 2 - Detection and characterization of monoclonal lymphocytic populations in clinical specimens by analysis of complementary RNA polymorphisms.

This assay can be performed on any clinical specimen, where a lymphocytic population is present and the DNA can be extracted and amplified by polymerase chain reaction. This is possible for biological materials, such as peripheral blood lymphocytes, DNA extracted from paraffin embedded histological specimens, lymph node or other biopsies.

Junctional DNA sequences of the T-cell receptor (gamma) genes and of the immunoglobulin heavy chain genes (CDR III) which are unique for an individual lymphocytic clone are amplified by polymerase chain reaction, using primers annealing to conserved sequences of these genes.

The 5' primer of the PCR reaction contains a T7 RNA polymerase promoter sequence (5' TAA TAC GAC TCA CTA TAG GGA G). This enables the transcription of the PCR amplified junctional sequences of T-cell receptor gamma genes and immunoglobulin heavy chain genes (CDR III regions) into RNA by a T7 RNA polymerase.

The synthesized complementary RNA of the hypervariable regions of the genes forms multiple conformations, that can be separated by a non-denaturing polyacrylamide gel electrophoresis.

In case of a predominance of a certain lymphocytic clone, different bands (specific band pattern) can be detected on the gel after staining with ethidium bromide. These unique patterns are formed when at least 1% of monoclonal cells are present in the specimen. The determination of the sensitivity of this method has been evaluated after mixing experiment with T-cell lymphocytic leukemia cells (CCRF-CEM cell lines) and polyclonal lymphocytes from peripheral blood of healthy individuals.

Materials and Methods

The following procedure with some modifications as described below was first used for the detection of point mutations in different genes. (See Experiment Number 1, Described hereinabove). The compounds and the reaction conditions for the whole procedure starting with a PCR product are as follows: Ribonucleotides 10X 2.5 ul

(10 mM ATP, CTP, GTP, UTP),

(Pharmacia, Piscataway, NJ)

Reaction buffer 10X 2.5 ul

(0.4 M Tris, pH 7.5, 0.12 M MgCL₂)

0.5 M Sermidine 0.1 ul

RNAse inhibitor (40,000 U/ml) 0.25 ul

(RNAsin, Promega, Madison, WI)

Dithiothreitol (1M) 0.25 ul

T7 RNA polymerase (60,000 U/ml) 0.6 ul

(Pharmacia)

PCR product 10-15 ul add DEPC (diethylpyrocarbonate) treated dH₂O to a final volume of 25 ul

**If not specified, no special supplier is preferred for the compounds and reactants.

A radioactive labelled ribonucleotide is used, if there is need to document the final reaction product by autoradiography. The mixture is incubated for one hour at 37ºC and the enzymatic process is stopped by adding 0.75 ul of 0.5 M EDTA. Ten to 20 ul of the reaction product are loaded on an 8% non-denaturing polyacrylamide gel with a cross-linkage of acrylamide/bisacrylamide of 19;1 and run at 15 Watt ("W") at 4ºC. The resolution of the bands is better on a polyacrylamide gels, but for convenience the non- gradient polyacrylamide gel is used for routine assays.

The gel is stained in an ethidium bromide solution and the bands are visualized on an UV screen.

Experiment Number 3 - Detection of a monoclonal lymphoid population by the amplification of T-cell receptor gamma junctional DNA sequences.

T-cell receptor gamma (TCR-gamma) genes rearrange in T-and preB-ALL. The junctional structures of V(N)J rearrangements are clonespecific and allow for the detection of a monoclonal population by different breakpoints in the rearrangement of different V and J genes and by the random nucleotide sequence of the inserted N-region. Junctional sequences of TCR-gamma were amplified in ALL by PCR using primers annealing to highly conserved regions of the variable and joining genes. The 5' primer annealing to the v genes contained a T7 RNA polymerase promoter sequence: The PCR product was transcribed into cRNA and electrophoreses on a 8-16% non-denaturing polyacrylamide gradient gel. Because of the clonespecific structure and the unique sequence of the highly variable N-region, multiple conformations of cRNA can be detected as distinct bands on the gel. This presents an idiotypic molecular "fingerprint" of the particular predominant clone. In contrast when cRNA is analyzed from a polyclonal lymphoid population, a brad smear on the gel is formed representing numerous conformations of different cRNA molecules. This method can detect as few as 1% monoclonal lymphoid cells. This novel and non-radioactive technique allows the detection of a clonal lymphoid population. Figure 7 shows the results of this experiment.

Experiment number 4 - Analysis of Immunoglobulin heavy chain gene rearrangements (e.g. CDR II regions)

The junctional sequences of IgH gene rearrangements were amplified by PCR and transcribed into complementary RNA. The reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel. See Figure 8 for results.

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Claims

What is claimed is:

1. A method to detect within a predetermined DNA sequence from a subject, the presence at a predefined position, a mutation, which comprises: a. obtaining from the subject a sample containing the predefined DNA sequence; b. amplifying the DNA sequence identified in step a; c. transcribing the amplified DNA to RNA; d. separating the RNA obtained in step c on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern; e. comparing the pattern of bands obtained in step d with the pattern of bands obtained by separating on a non-denaturing gel RNA transcribed from a sample known to contain the predetermined DNA sequence without the mutation at the predefined position; and f. detecting any differences in the patterns of step e, the presence of a difference in the patterns indicating the presence of a mutation at the predefined position of the predetermined DNA.

2. The method of claim 1, which further comprises labeling the RNA obtained in step c prior to gel separation.

3. The method of claim 2, wherein the RNA is labeled with a radioisotope or fluorescent agent.

4. The method of claim 1, wherein the DNA sequence is amplified by enzymatic amplification.

5. The method of claim 1, wherein the suitable DNA sample is obtained from paraffin-embedded material.

6. The method of claim 1, wherein the mutation is a point mutation.

7. The method of claim 1, wherein the DNA is genomic DNA, single-stranded DNA, double-stranded DNA, plasmid DNA, bacterial DNA, parasitic DNA or viral DNA.

8. The method of claim 1, wherein the non-denaturing gel is a polyacrylamide gel (PAGE).

9. The method of claim 1, wherein the subject is an animal.

10. The method of claim 9, wherein the animal is a mammal.

11. The method of claim 10, wherein the mammal is a human.

12. A method for detecting in a monoclonal lymphoid population of cells in a subject, the presence at a predetermined position of a clonespecific DNA sequence, which comprises: a. obtaining from the subject a DNA sample containing the predetermined position; b. amplifying the DNA obtained in step a; c. transcribing the amplified DNA to RNA; d. separating the RNA obtained in step c on a non-denaturing gel so as to separate the RNA into one or more bands having a defined pattern; e. comparing the pattern of bands obtained by separation on non-denatureing gel to RNA transcribed from a DNA sample known to contain the predetermined position without the clonespecific sequence; and f. detecting any differences in the patterns of step e, the presence of a difference in the patterns indicating the presence of the monoclonal lymphoid population of cells.

13. The method of claim 12, wherein the monoclonal lymphoid population of cells are T-cells.

14. The method of claim 13, the predetermined position is the DNA sequence corresponding to the

T-cell receptor gamma gene.

15. The method of claim 13, wherein the predetermined position is the DNA sequence corresponding to the T-cell gamma junctional gene.

16. The method of claim 12, wherein the monoclonal lymphoid population of cells are B cells.

17. The method of claim 16, wherein the predetermined position is the DNA sequence corresponding to the variable DNA sequences in the junctional region of immunoglobulin genes.

18. The method of claim 16, wherein the variable DNA sequence corresponds to CDR I, CDR II or CDR III or IgH.

19. The method of claim 12, which further comprises labeling the RNA obtained in step c prior to gel separation.

20. The method of claim 19, wherein the RNA is labeled with a radioisotope or fluorescent agent.

21. The method of claim 12, wherein the DNA sequence is amplified by enzymatic amplification.

22. The method of claim 7, wherein the DNA is obtained from lymphocytic paraffin-embedded material; lymph node specimens; and lymphocytic biopsies.

23. The method of claim 12, wherein the subject is an animal.

24. The method of claim 23, wherein the animal is a mammal.

25. The method of claim 24, wherein the mammal is a human patient.

26. The method of claim 1 or 12, which further comprises staining the gels of steps d and e with ethidium bromide.