WO2003031646A1 - Multiple genetic marker selection and amplification - Google Patents

Multiple genetic marker selection and amplification Download PDF

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WO2003031646A1
WO2003031646A1 PCT/AU2002/001388 AU0201388W WO03031646A1 WO 2003031646 A1 WO2003031646 A1 WO 2003031646A1 AU 0201388 W AU0201388 W AU 0201388W WO 03031646 A1 WO03031646 A1 WO 03031646A1
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nucleic acid
pcr
dna
genetic
cell
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PCT/AU2002/001388
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French (fr)
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Ian Findlay
Paul Lawrence Matthews
Brendan Khalid Mulcahy
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The University Of Queensland
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Priority claimed from AUPR8234A external-priority patent/AUPR823401A0/en
Priority claimed from AUPR8235A external-priority patent/AUPR823501A0/en
Application filed by The University Of Queensland filed Critical The University Of Queensland
Priority to EP02800523A priority Critical patent/EP1442139A4/en
Publication of WO2003031646A1 publication Critical patent/WO2003031646A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6858Allele-specific amplification

Definitions

  • TITLE MULTIPLE GENETIC MARKER SELECTION AND AMPLIFICATION FIELD OF THE INVENTION relates to selection and amplification of genetic markers for genetic analysis. More particularly, this invention relates to selection of genetic markers and primers to facilitate multiplex PCR amplification from limiting amounts of nucleic acid.
  • the methods of the invention are generally applicable to improved genetic testing methods including but limited to diagnostics and screening, for example prenatal diagnostic testing, fetal sex determination and genetic identification, such as by DNA fingerprinting, in organisms including but not limited to bacteria, plants, humans and other animals where available nucleic acid is limiting.
  • the nucleic acid amplification method of the invention is also applicable to by genetic identification of degraded, old, ancient, difficult and or low-abundance samples that have hitherto been difficult to amplify, detect and/or identify.
  • Conventional genetic analysis generally consists of a single test on large amounts of sample, for example diagnosis of ⁇ F508 (major cystic fibrosis mutation) from DNA extracted from a relatively large volume (5 mL) of blood.
  • PCR multiplex polymerase chain reaction
  • STRs polymorphic short tandem repeats
  • Quantitative PCR accurately determines the amount of PCR product from each allele permitting the ratio of product quantity between alleles to be calculated thus determining aneuploidy status. Even though quantitative PCR was first described in the early 1990s, there are few reports in clinical prenatal diagnosis (Adinolfi et al., 1995.
  • MF-PCR multiplex fluorescent PCR
  • Trisomic samples produce either a triallelic signal (three peaks of similar size) seen in Panel 1, or a diallelic or double-dose signal (two peaks, one of which is approximately twice the size of the other) seen in Panel 2.
  • Disomic samples produce a heterozygous signal (double peaks of similar size) seen in Panel 3.
  • Homozygous signal showing a single peak (not shown) are regarded as uninformative, as they can be obtained from both disomic and trisomic samples.
  • Diallelic signals are partially informative with triallelic signals being the most informative.
  • Multiplex fluorescent PCR provides two main advantages. Firstly, the multiplex system provides multiple diagnoses, using either linked markers to confirm results or to expand the scope of the test to multiple chromosomes or perform other diagnoses. Secondly, the use of fluorescent markers significantly increases the threshold of detection almost 1000 fold (Findlay et al, 1995, Human Reproduction 10 1005). Generally, however, it has not been possible to use these techniques to diagnose trisomies at or near the single cell level (Findlay, 1998, supra; Findlay et al, 1998a & 1998c, supra; Findlay et al, 1999, Journal of Assisted Reproduction and Genetics 16 199-206).
  • Fluorescent, multiplex PCR has been shown a reliable and accurate method for determining sex (Salido et al, 1992, Am. J Human genetics 50 303; Findlay et al, 1994a, Human Reproduction, 9 23; Findlay et al., 1994b, Advances in Gene Technology: Molecular Biology and Human Genetic Disease. Vol 5, page 62.
  • the present inventors have sought to improve the quality of information obtainable from amplification of multiple genetic markers in applications relating to genetic testing and identification, such as in prenatal diagnosis, genetic disease screening and testing and DNA fingerprinting, such as in forensics.
  • a problem associated with improving the performance of diagnostic nucleic acid sequence amplification, particularly from limiting amounts of nucleic acid template, is that the more genetic markers are amplified in a multiplex reaction, the more compromised is the quality of the information obtained from the reaction.
  • the present inventors have improved the selection of genetic markers that can be amplified in combination, the number of genetic markers that can be amplified in combination and the efficiency of amplification from limiting amounts of nucleic acid template
  • the present invention provides a method of selecting a plurality of genetic markers as targets for nucleic acid sequence amplification, said method including the step of selecting each of said plurality of genetic markers according to a heterozygosity index, wherein said heterozygosity index is 0.5 or greater.
  • said heterozygosity index is 0.7 or greater.
  • said heterozygosity index is 0.9 or greater.
  • the invention provides a method of producing one or more primers for amplification of each of a plurality of genetic markers selected according to the first aspect of the invention, said method including the step of selecting a nucleotide sequence for each of said one or more primers so that upon amplification of said genetic marker using said one or more primers, a resultant amplification product has a molecular size in the range 50-3000 bp.
  • the molecular size is in the range 50-1000 bp.
  • the molecular size is in the range 80-500 bp. Even more preferably the molecular size is in the range 100-400 bp.
  • Primers constructed according to this aspect may be degenerate or non- degenerate as is well understood in the art.
  • the invention provides a method of nucleic acid sequence amplification including the step of using a nucleic acid sequence amplification technique and at least nine primer pairs in combination to amplify a plurality of respective genetic markers from a limiting amount of nucleic acid sample.
  • At least ten, eleven, twelve, thirteen, fourteen, fifteen and sixteen primer pairs are used to amplify said respective genetic markers.
  • each said primer pair amplifies a respective genetic marker.
  • nucleic acid sequence amplification is performed using PCR.
  • PCR is fluorescent multiplex PCR.
  • Table 1 provides non-limiting examples of primers and resultant amplification fragment sizes applicable to each genetic marker.
  • a reference to the DYS14 marker is Lo et al, 1993, Hum. Genet. 90 483.
  • Primers are attributed SEQ ID NOS:l-92 in order of appearance in Table 1.
  • TABLE 2 Non-limiting examples of fluorescently-labeled primers and corresponding genetic markers applicable to multiple genetic diagnoses.
  • TABLE 3 Non-limiting examples of fluorescently-labeled primers and corresponding genetic markers applicable to DNA fingerprinting.
  • TABLE 4 Comparison of FISH, PRTNS and fluorescent multiplex PCR techniques for preimplantation genetic diagnosis (PGD).
  • TABLE 5 Single cell DNA fingerprinting improvements. The new method is that described herein; the published method is that described in Findlay et al, 1997, Nature 389 355-356. ! Full profiles provide highest possible specificity. However as more STR markers are added it becomes more likely that one or more will fail or be compromised.
  • Table 6a shows allele sizes obtained for each marker. It can be seen that the genetic identification allele sizes for twin 1 are identical to that of twin 2 thus indicating that the twins are identical twins.
  • Table 6b demonstrates maternal or paternal derivation of each allele thus indicating maternity and paternity.
  • FIG. 1 Quantitative fluorescent PCR in prenatal diagnosis.
  • Panel two shows 2: 1 ratio or diallelic signal.
  • FIG. 2 Multiplex fluorescent PCR from a single cell sample with nine out of nine genetic markers present.
  • FIG. 3 Limited nucleic acid template sample subjected to eleven (11) primer set multiplex PCR. In this example 10 of 11 markers were amplified.
  • FIG. 4 Eleven (11) primer set multiplex PCR on single diploid cell. 11 of 11 markers were amplified
  • FIG. 5 Eleven (11) primer set multiplex PCR on single sperm (haploid cell).
  • FIG. 6 Sixteen (16) primer multiplex PCR on single diploid cell. 16 of 16 markers amplified
  • FIG. 7 Genetic identification of single fetal cell isolated from PAP smears using nine (9) primer pairs. 9 of 9 markers amplified. Maternal genetic identification is also shown to demonstrate that both fetal signal and maternal signals share common alleles (indicating maternity), but the fetal cell has inherited other alleles from a paternal source, consistent with Mendelian inheritance.
  • FIG. 8 Genetic identification of single fetal cell isolated from PAP smear using eleven (11) primer pairs.
  • FIG. 9 Nine (9) primer pair multiplex PCR demonstrating twin heterozygosity using limited amount of amniotic fluid. Results indicate both twins identical.
  • FIG. 10 Nine (9) primer pair multiplex PCR demonstrating twin heterozygosity using limited amount of amniotic fluid. Results indicate both twins non-identical.
  • FIG. 11 Multiplex fluorescent PCR from a single cell sample with nine (9) out of nine (9) genetic markers present.
  • FIG. 12 DNA fingerprint obtained from hairshaft using eleven (11) primer fluorescent multiplex PCR.
  • FIG. 13 Ten (10) primer pair fluorescent multiplex PCR demonstrating genetic diagnosis of trisomy status from limited amount of amniotic fluid.
  • FIG. 14 Ten (10) primer pair multiplex demonstrating simultaneous diagnosis of single-gene defect (cystic fibrosis), sex, trisomy status and genetic identification. DETAILED DESCRIPTION OF THE INVENTION
  • the invention described herein relates to nucleic acid sequence amplification of multiple genetic markers, and methods of selecting genetic markers to improve the efficiency of marker amplification.
  • Heterozygosity is defined as the presence of different alleles of a gene at one or more loci. Heterozygosity occurs when a diploid organism or cell has inherited different alleles at a particular locus from each parent. Heterozygosity index is a measure of the likelihood of marker alleles being different within individuals i.e. having two alleles rather than one. For example alleles from markers with low heterozygosity are more likely to be identical or be homozygous within an individual or population.
  • Markers with higher heterozygosities are more likely to provide triallelic (most informative) results (see Figure 1), if the sample is trisomic. It is therefore necessary to choose markers with as high heterozygosity (dp) values as possible.
  • Fragment size The optimal fragment size window is between 100-400bp although 80bp to 500bp, 50bp to lOOObp or even 50 bp to 3000 bp can be used. Fluorescent systems for fragment detection have increasingly limited detection when fragment size is less than 80bp due to interference from primer dimer. Fragment sizes that are large, (e.g. greater than 500bp), even though they may not accurately be sized can still be used to identify multiple peaks and triallelic results. In general the larger the fragment size the more time it takes for results to be obtained. As most diagnostic laboratories require results as quickly as possible, smaller fragments would therefore be most preferred. Additional considerations include:-
  • chromosome It is necessary to choose a marker that will accurately reflect the test being performed. For example, if one is attempting to determine the number of copies of chromosome 21, a marker on chromosome 21 is most likely to be the most appropriate.
  • Fluorescent labeling Using fluorescent labeled primers to combine markers in a multiplex with markers of similar or overlapping fragment ranges. The choice of fluorescent label is very important since marker allele sets can overlap with each other. Overlap with another marker would make the marker of limited value since each marker may then be indistinguishable from the other. For example if one marker was heterozygous and the other homozygous this would show as a triallelic response incorrectly indicating a trisomy.
  • the marker When marker size sets do overlap, the marker could be labelled with a differently coloured fluorochrome thus allowing identification of each marker.
  • the present invention therefore provides a substantial improvement in the efficiency of genetic marker selection such as for the purposes of selecting STRs that allow PCR amplification of multiple genetic markers for applications including genetic identification (for example, human embryo identification and forensics), genetic diagnosis and screening (for example, pre-implantation genetic diagnosis after IVF and from fetal cells obtained from cervical smears, CVS or amniocentesis), although without limitation thereto.
  • the present invention provides multiplex PCR amplification using at least nine primer pairs to amplify discrete genetic markers from limiting amounts of nucleic acid sample in a highly efficient manner. For example, nine (9) informative genetic markers were successfully amplified in 69 of 69 multiplex amplifications using nine (9) primer pairs and nucleic acid samples from single buccal cells. The present invention also contemplates amplification of up to and in excess of sixteen (16) genetic markers as will be described in more detail hereinafter.
  • genetic analysis and genetic diagnosis are used interchangeably and broadly cover detection, analysis, identification and/or characterization of genetic material and includes and encompasses terms such as, but not limited to, genetic identification, genetic diagnosis, genetic screening, genotyping and DNA fingerprinting which are variously used throughout this specification.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native or recombinant form.
  • nucleic acid designates single-or double-stranded mRNA, RNA, cRNA, RNAi and DNA, said DNA inclusive of cDNA and genomic DNA.
  • a “polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides.
  • genetic marker or “marker' ' ' is meant any locus or region of a genome.
  • the genetic marker may be a coding or non-coding region of a genome.
  • genetic markers may be coding regions of genes, non-coding regions of genes such as introns or promoters, or intervening sequences between genes such as those that include tandem repeat sequences, for example satellites, microsatellites, short tandem repeats (STRs) and minisatellites, although without limitation thereto.
  • a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
  • Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999); strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • NASBA nucleic acid sequence-based amplification
  • Q- ⁇ replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395.
  • multiplex amplification or “multiplex PCR” refers to amplification of a plurality of genetic markers in a single amplification reaction.
  • the invention provides "fluorescent PCR”.
  • This system uses fluorescent primers and an automated analyser such as a DNA sequencer to detect PCR product (Tracy & Mulcahy, 1991, Biotechniques 11 68-75). Fluorescent PCR has improved both the accuracy and sensitivity of PCR for genotyping (Ziegle et al, 1992, Genomics, 14 1026-1031; Kimpton et al, 1993, PCR Methods and Applications 3 13-22).
  • fluorescent amplification products are electrophoresed using gel or capillary systems and pass a scanning laser beam, which induces the tagged amplification product to fluoresce.
  • the DNA sequencer combined with appropriate software is generally known as a "Genescanner”. Stored data can then be analysed to provide product sizes and the relative amount of amplification product in each sample.
  • a preferred nucleic acid sequence amplification technique is PCR.
  • an "amplification product' refers to a nucleic acid product generated by a nucleic acid amplification technique.
  • a “primer” is usually a single-stranded oligonucleotide, preferably having 12-
  • nucleotides which, for example, is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent
  • Non-limiting examples of primers that may be used in combination are primers capable of amplifying genetic markers (STR loci) index listed in Table 1 .
  • each said primer may be fluorescently-labeled to produce a fluorescently-labeled primer pair.
  • Fluorescent labels are well known in the art and include but are not limited to TET, FAM, HEX as for example described in Table 2.
  • fluorescent labels potentially useful according to the invention include but are not limited to CyDyesTM such as Cy2, Cy3, C3.5, and Cy5.
  • Non-limiting examples of fluorescent-labeled primers are set forth in Table 2.
  • Primer synthesis and incorporation of fluorescent labels are well known in the art and labeled, synthetic primers are readily available from commercial sources.
  • an example of primer synthesis methodology is provided in Chapter 2..11 of Current Protocols in Molecular Biology Ausubel et al. Eds (John Wiley & Sons, NY, 1996-2001). It will be appreciated by the skilled that the present invention is particularly suited to selection of genetic markers and corresponding primers for the purposes of nucleic acid sequence amplification from limiting amounts of nucleic acid.
  • a "limiting amount of nucleic acid” is an amount of nucleic acid used in a nucleic acid sequence amplification reaction less than 1 ng, preferably less than 500 pg, more preferably less than 200 pg, even more preferably less than 50 pg and in particular embodiments, about 3-6 pg.
  • a limiting amount of nucleic acid may also relate to the number of cells containing the nucleic acid sample used for amplification.
  • the limiting amount of target nucleic acid is obtained from less than
  • 200 cells more preferably from less than 100 cells, more preferably from less than 50 cells and even more preferably from less than 20 cells.
  • the limiting amount of nucleic acid is isolated from no more than ten cells, or from a single cell.
  • the present inventors have performed multiplex PCR amplification from a single, haploid sperm cell which comprises about 3-6 pg of DNA.
  • nucleic acid other than DNA, preferably the nucleic acid is DNA.
  • the nucleic acid is genomic DNA.
  • suitable sources of cells from which DNA may be obtained include, but are not limited to, buccal cells, sperm cells, hair follicle cells, skin cells, epithelial cells, nucleated cells circulating in blood, embryonic cells, fetal cells such as obtained from fetal blood, CVS or amniocentesis samples or cervical (PAP) smears, corneal cells, cell or tissue biopsies, or any other cells or tissues from which genetic material can be obtained.
  • the invention is applicable to genetic analysis from small numbers of cells such as for the purposes of prenatal diagnostic testing or screening, fetal sex determination and genetic identification by DNA fingerprinting.
  • Cellular sources such as described above may be of any organism inclusive of plants, bacteria and animals.
  • Preferred sources of nucleic acids are mammals, preferably humans.
  • the invention also contemplates genetic analysis of non-human samples such as from cows, sheep, horses, pigs and the like, although without limitation thereto.
  • nucleic acids may be obtained from non-cellular sources such as viruses.
  • nucleic acids are not necessarily directly obtained from their cellular or non-cellular source organism.
  • nucleic acids may be obtained from objects such as paper, documents, clothing, bedding, motor vehicles, physical fingerprints, weapons, furniture and building fixtures and a variety of substrates such as biological fluids and ink although without limitation thereto, such as for the purposes of genetic identification.
  • cells or nucleic acid material may or may not require isolation by techniques as are well known in the art. These include but are not limited to densitometric separation such as by gradient centrifugation through media such as MetrizamideTM, FicollTM and PercollTM, biochips, micro-manipulation, pulse field separation, differential lysis and antibody- mediated isolation such as by panning, magnetic bead separation or fiow-cytometric (FACS) sorting.
  • densitometric separation such as by gradient centrifugation through media such as MetrizamideTM, FicollTM and PercollTM, biochips, micro-manipulation, pulse field separation, differential lysis and antibody- mediated isolation such as by panning, magnetic bead separation or fiow-cytometric (FACS) sorting.
  • FACS fiow-cytometric
  • Cytometry 7 536 where the use of centrifugal separation of cells in sputum specimens is described.
  • the invention provides a method of prenatal genetic diagnosis of a fetus wherein a nucleic acid sample for amplification is obtained from one or more fetal cells isolated from a pregnant individual (i.e the mother).
  • a nucleic acid sample for amplification is obtained from one or more fetal cells isolated from a pregnant individual (i.e the mother).
  • fetal includes embryonic cells at any developmental stage from any species.
  • Fetal cells may be isolated by any cell isolation method.
  • Said one or more fetal cells may be isolated from any pregnant mammal.
  • said one or more fetal cells are isolated from a pregnant human.
  • prenatal diagnosis is by invasive procedures such as either chorionic villus sampling (CVS) in the late 1 st trimester or amniocentesis in the 2 nd trimester of pregnancy.
  • CVS chorionic villus sampling
  • amniocentesis in the 2 nd trimester of pregnancy.
  • CVS chorionic villus sampling
  • amniocentesis or even fetal blood sampling, may be necessary.
  • a rapid, less-invasive and low cost method of prenatal diagnosis involves genetic diagnosis from fetal cells shed into the cervical sump at 6-20 weeks of gestation.
  • said fetal cells are present in a maternal cavity, such as the uterus, or endocervical canal sample, particularly a transcervical sample.
  • Methods of isolating fetal cells include but are not limited to cervical cotton swab, cytobrush, aspiration of cervical mucus, lavage of the endocervical canal and uterine lavage.
  • Samples can be obtained from transcervical aspiration of mucus from just above the internal os or the lower uterine cavity.
  • Another isolation method which may be used is lavage which is generally conducted with a saline wash, but other isotonic solutions are suitable.
  • endocervical lavage with 5- 10ml or intrauterine lavage with 10-20 ml saline provides sufficient fetal cells upon separation from maternal cells.
  • the sample may be collected using a combination of methods.
  • cell samples are isolated from a female human in the first trimester of pregnancy or when the fetus is between 6 to 20 weeks gestation.
  • clumps of cells are preferably treated to obtain a suspension of single cells.
  • the clumps may be separated by techniques known to a skilled person, such as enzymatic, chemical or mechanical separation.
  • enzymatic separation may utilise protease or trypsin.
  • Chemical separation may utilise acetyl cysteine and mechanical separation may involve gentle teasing, aspiration or micromanipulation.
  • the number of fetal cells in the sample varies depending on factors including the age of the fetus, method of sampling, number and frequency of samplings, the volume of washing in each lavage where lavage is used and the volume aspirated.
  • Maternal uterine cavity or endocervical canal samples typically contain at least two main types of nucleated fetal cells: cytotrophoblasts and syncytiotrophoblasts cells.
  • Fetal cells can be isolated either by selecting fetal cells from maternal cells (positive selection) or isolating the maternal cells from the fetal cells (negative selection) or most preferably a combination of both.
  • the nucleated fetal cells are retained in the purified sample.
  • one method to isolate fetal cells is micromanipulation.
  • cell suspensions containing an individual cell per a preselected volume of suspension medium can be prepared by limiting dilution. Drops containing individual cells can placed in suitable container (e.g. 96 well plates) and examined visually with a fluorescent microscope to identify single- labelled (or unlabelled) cells.
  • analysis can be performed using a single, identified fetal cell.
  • ways can be envisaged of identifying monozygosity (indicative of the presence of a monogenic disease) in a mixed cell population containing minimal fetal material including as few as one fetal cell in 100 cells.
  • the separated cells can be washed twice in a physiologic buffer and resuspended in an appropriate medium for any subsequent analysis to be performed on the cells.
  • the fetal cells can be used in the same manner as fetal cells obtained by other methods such as amniocentesis and chorionic villus biopsy.
  • the cells can be used as a source of DNA for analysis of the fetal alleles, as by polymerase chain amplification for example.
  • PCR analysis methods may be used to detect, for example, fetal sex, beta thalassemia, phenylketonuria (PKU), and Duchennes muscular dystrophy without limitation thereto.
  • the cells can be cultured in a similar manner as material biopsied for karyotyping analyses.
  • the incubation period may be significantly shortened if a DNA content of greater than or equal to 2C is used as a selection criterion.
  • the present invention provides a method of prenatal analysis using nucleic acids isolated from fetal cells isolated by but not limited to invasive procedures such as from fetal blood, amniocentesis or CVS.
  • An alternative diagnostic method is quantitative PCR using polymorphic short tandem repeats (STRs) to accurately determine PCR product ratio from each allele and thus aneuploidy status.
  • STRs polymorphic short tandem repeats
  • Limited marker sets with limited diagnostic capability which, for example, generally only determine aneuploidy and/or sex.
  • the present invention in one embodiment provides improved multiplex nucleic acid sequence amplification on limited samples to overcome these difficulties.
  • This invention in one embodiment will significantly improve diagnostic confidence, capability as well as reduce cost and time.
  • cell samples are isolated from a female human in the first trimester of pregnancy or when the fetus is between 6 to 20 weeks gestation and may consist of amniotic fluid or samples from the chorionic villi.
  • the number of fetal cells in the sample varies depending on factors including the age of the fetus, method of sampling, skill of operator, number and frequency of samplings, the amount of sample obtained in each procedure and the volume aspirated.
  • Amplification methods such as PCR analysis may be used to detect, for example, fetal sex, beta thalassemia, phenylketonuria (PKU), and Duchennes muscular dystrophy without limitation thereto.
  • the present invention relates to genetic analysis or genetic identification by "DNA profiling" or commonly known as DNA fingerprinting of samples.
  • cellular and/or non-cellular nucleic acid samples can be obtained from a variety of sources including but not limited to forensic samples (such as clothing, bedding, motor vehicles, physical fingerprints, weapons, furniture and building fixtures and a variety of substrates such as biological fluids and ink), documents or other substrates such as ink or paper and ink derived therefrom, archaeological or other old or ancient samples, samples obtained for the purposes of personal identification, biological samples or clinical samples such as used for genetic identification, testing, screening and/or diagnosis of genetic diseases, sexing and detection of chromosomal abnormalities.
  • DNA profiling is an extremely powerful method for forensic identification with current prior art achieving power of discrimination in excess of 1 in 10 billion.
  • STR profiling systems have been applied to low cell samples such as cigarette butts (Torre and Gino, 1996, J Forensic Sci. 41 658-9) and from cells left on pens, car keys, etc (van
  • a particular problem applies in rape cases, particularly multiple rape where semen from multiple sources are present.
  • Conventional forensic analysis requires a clean uncontaminated sample to obtain a DNA fingerprint but, as the semen may be a mixed sample (for example, from each assailant, or assailant and male partner), definitive DNA fingerprints are usually not possible. This leads to a failed forensic test, with the result that there may be insufficient evidence for the prosecution or defence.
  • the present invention provides an improved method whereby genetic identification by DNA fingerprinting can now be obtained from small samples and or single cells such as single sperm to determine their origin and thus identify each assailant.
  • single cells may be obtained from samples which have too few cells for conventional profiling; from samples contaminated by blood or other cell types; from archived cases; old previously solved or unsolved cases; and from physical fingerprints.
  • the single cell DNA fingerprint test described here could be applied for genetic identification to a wide variety of samples and sample types including but not limited to smudged physical fingerprints, single flakes of dandruff, as well as small samples left on weapons, vehicles and other objects.
  • IVF success rates have remained relatively constant at only ⁇ 10-20% per embryo transferred. This may be because a sizeable number of human embryos are chromosomally or otherwise abnormal and therefore unable to implant, or form or maintain a pregnancy. It has been possible since 1990 to diagnose genetic defects from single embryonic cells removed from embryos (preimplantation genetic diagnosis (PGD) also alternatively known as preimplantation diagnosis (PID)). There are three main applications for PGD: sex, single gene defects and aneuploidy e.g. trisomy diagnosis. In general FISH (fluorescent in situ hybridisation) is used for sex or aneuploidies and PCR for single gene defects.
  • FISH fluorescent in situ hybridisation
  • Single cell fluorescent PCR has previously shown to be highly reliable (97%), highly accurate (97%), rapid (6hrs) and wide ranging (simultaneous diagnoses of sex, single gene defects and trisomies) (Findlay et al, 1995, Human Reproduction 10 1609- 1618). However such testing has been limited to a limited number (upto and including 8) of markers, which limits use.
  • Single cell fluorescent PCR can also determine a DNA fingerprint from a single cell therefore minimising the risk of misdiagnosis due to contamination (Findlay et al, 1995, Human Reproduction 10 1005-1013; Findlay, 1996, Human Reproduction Update 2 137-152; Findlay et al, 1997, Nature 389 355-356; Henderson et al, 2001, Cornea 20 400-403). Again such testing has been limited to a limited number (up to and including 8) of markers, which severely limits use in genetic identification particularly since a major source of contamination is parental DNA which share common alleles with the embryonic cell thus significantly decreasing specificity of discrimination of the DNA fingerprint. Fluorescent PCR can be favourably compared to other techniques as shown in Table 4.
  • DNA fingerprinting of embryonic cells allows individual embryos to can be genetically "tracked” from the 6-8-cell stage to birth and beyond. This makes it possible to determine which pregnancy resulted from which embryo.
  • the present invention allows DNA fingerprinting to be performed on single cells with a specificity greater than 10 billion to 1, far in excess of any other single cell genotyping system and far in excess of prior art (Findlay et al, 1997, Nature 389 355-356) single cell DNA fingerprinting at -100 million to 1.
  • this embodiment of the present invention provides but is not limited to: a clinical tool that provides a quality control mechanism; patient reassurance that correct embryos are identified for transfer; determination of separate pregnancy rates in multiple embryo transfer e.g.
  • the method of the invention may be used to provide accurate and absolute correlation of embryo quality with pregnancy and might be used to accurately compare differing culture conditions. For example embryos cultured in two different media can be transferred to the same woman and an accurate pregnancy rate per media derived. Patient reassurance is also improved by the PCR method of the invention by confirming that embryos transferred are genetically derived from parents.
  • the invention may be used for genetic analysis such as PGD or prenatal diagnosis or screening from non-human sources.
  • Such non-limiting examples include PGD or genetic screening of an increased number of a wide variety of genetic traits to improve qualities from domestic animals such as cattle.
  • VNTRs variable number tandem repeats
  • the number of repeats is highly variable among individuals and heterozygosity is high (i.e. the number of repeats at the locus is usually different on the two pairs of chromosomes of one individual). Analysing the number of repeats at one or more such loci provides a highly sensitive measure of individual identity and is the preferred technique for forensic DNA typing as means of genetic identification.
  • Tandem repetitive sequences are classified into three major groups: 1. Satellites are very highly repetitive with repeat lengths of one to several thousand base pairs. These sequences typically are organized as large (up to 100 million bp) clusters in the heterochromatic regions of chromosomes, near centrosomes and telomeres; these are also found abundantly on the Y chromosome. 2. Minisatellites are moderately repetitive, tandemly repeated arrays of moderately-sized (9 to 100 bp, but usually about 15 bp) repeats, generally involving mean array lengths of 0.5 to 30 kb. They are found in euchromatic regions of the genome of vertebrates, fungi and plants and are highly variable in array size. 3.
  • Microsatellites are moderately repetitive, and composed of arrays of short (2-6 bp) repeats found in vertebrate, insect and plant genomes.
  • the human genome contains at least 30,000 microsatellite loci located in euchromatin. Copy numbers are characteristically variable within a population, typically with mean array sizes on the order of 10 to 100.
  • Microsatellite loci are highly polymorphic sequences elements in the human genome, and delineating the repeat lengths of these loci is the basis of most DNA typing systems used in forensic medicine.
  • Heterozygosity is defined as the presence of different alleles of a gene at one or more loci. Heterozygosity occurs when a diploid organism or cell has inherited different alleles at a particular locus from each parent. Both cases result in mixtures of DNA sequences that have important applications in fields such as forensics, pathology, genetic diagnosis, and evolutionary genetics.
  • Genetic marker and primer selection Polymorphism in a population is due to the existence of different genetic variants. The basis of variation is thus the number of polymorphic loci together with the number of alleles and their frequency distributions in a population. Based on this concept, markers in Table 1 were checked for the number of different alleles and genetic diversity, both by determining allele frequencies and from data provided publicly through public databases such as GenBank. Markers with higher heterozygosity rates (highly variable) are selected in preference.
  • PCR protocol for sexing, chromosome 21, 18 and 13 detection A limited number of cells were isolated from amniotic fluid.
  • a mastermix containing the reagents required for the PCR is made up under aseptic conditions.
  • the mastermix contains enough reagents for a number of 25 ⁇ l reactions.
  • the primers together with an indication of fluorescent labels for each primer are shown in Table 2, and the composition of the mastermix, per reaction, is as follows:- Reagents Amoun ⁇ ⁇ l
  • the mastermix is mixed thoroughly and added to template, or if using a plate, the mastermix is aliquoted and template added to it.
  • the tubes/plate was placed on a thermal cycler and subjected to the following PCR program:-
  • Lysis is carried out by adding l ⁇ l of Lysis Buffer (200mM KOH, 50mM DTT) to the cell or cells.
  • the cell mixture is spun down and is ready for PCR or stored at -80°C until needed. PCR protocol
  • a mastermix containing the reagents required for the PCR is made up under aseptic conditions.
  • the mastermix contains enough reagents for a number of 25 ⁇ l reactions.
  • the primers together with an indication of fluorescent labels for each primer are shown in Table 3 and the composition of the mastermix, per reaction, is as follows:- Reagents Amount/ ⁇ l
  • the mastermix is mixed thoroughly and added to l ⁇ l of template, or if using a plate, the mastermix is aliquoted and the template is added to it.
  • the tubes/plate is placed on a thermal cycler and subjected to the following program:- 1. 95°C for 14 minutes
  • the PCR uses no oil overlay, as the heated lid of the PCR is sufficient.
  • the PCR is taken off the block and stored at 4°C until required for electrophoresis.
  • Multiplex PCR from a single cell sample with nine out of nine genetic markers present Single cells were isolated by micro-manipulation from buccal cell samples
  • Genetic markers are AMEL (1), D13S631 (2), D13S258 (3), D18S851 (4), D18S391 (5), DYS14 (6), D21S11 (7), D21S1411 (8) & D21S1412 (9) as shown in Figure 2.
  • Primer concentrations were: - AMEL 3.5 pmole
  • PCR cycling programme for the PCR in Figure 2 was: a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at 4°C.
  • the following PCR conditions are used for all single-cell and low copy analysis unless stated differently.
  • Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix consisted of 2 units taq polymerase (Amplitaq Gold (Applied Biosystems)); lx PCR buffer and 1.5mM Magnesium Chloride (provided with the Amplitaq Gold); dNTP's (0.2mM concentration) and fluorescent and non- fluorescent primers at varying concentrations. The master-mix was made up to 24 ⁇ l using MiUi-Q sterilised water.
  • Single cells were isolated using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - l ⁇ l of PBS drawn with the single cell.
  • PBS Phosphate buffered saline
  • Magnesium Magnesium
  • DNA analysis was performed using DNA sequencers such as ABI 377 or Megabace 1000 using standard protocols.
  • FIG. 3 shows a low copy number sample subjected to 10 primer set multiplex. Single cells were isolated by micro-manipulation from buccal cell samples.
  • the PCR amplified genetic markers are AMEL (1), D13S631 (2), D18S851 (3), DYS14 (4), D18S391 (5), D13S317 (6), D21S11 (7), D13S258 (8), D18S51 (9), D21S1412 (10).
  • PCR cycling programme for PCR for Figure 3 - a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at4°C.
  • Primer concentrations were: -
  • Figure 4 shows an electrophorogram of eleven genetic markers AMEL, THO, D21S11, D18S51, VWA, FGA, D3S1358, D5S818, D7S820, CSF and TPOX amplified from DNA template obtained from a single cell isolated from a buccal cell sample.
  • Single cell samples are added to lul of lysis buffer (200mM KOH/50mM DTT), heated to 65°C for 10 minutes, lul of neutralising buffer (300mM KCl/900mM Tris- HC1, ph8.3/200mM HC1) was then added.
  • lysis buffer 200mM KOH/50mM DTT
  • neutralising buffer 300mM KCl/900mM Tris- HC1, ph8.3/200mM HC1
  • PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride was added. dNTP's were added to reach 0.2mM concentration. Primers were added as described for each individual case. The master- mix was made up to 24 ⁇ l using Milli-Q sterilised water.
  • the electropherogram shown in Figure 5 shows the results of multiplex PCR amplification from DNA template obtained from a single sperm cell.
  • Primer concentrations were:- Marker pmol/rxn
  • the single cells were subjected to lysis prior to PCR. Each single cell had 5 ⁇ l of
  • FIG. 6 shows an electropherogram that demonstrates successful amplification of sixteen (16) genetic markers.
  • 16 of 16 markers amplified successfully.
  • Single cells obtained from buccal cell samples were subjected to lysis prior to PCR. Each single cell had 5 ⁇ l of 0.624 mg/ml Proteinase K. The single cells were then subjected to the following heating program: a) 50°C for 30 minutes b) 95°C for 15 minutes
  • PAP smear In this example 9 of 9 markers amplified successfully. PCR conditions were:- a) 94°C for 2 minutes b) 94°C for 30 seconds c) 57°C for 60 seconds d) 68°C for 30 seconds e) Go to (b), 45 times f) 72°C for 10 minutes g) Hold at 4°C
  • Isolated single cell samples were fixed then lysed by alkaline lysis using standard techniques before PCR processing.
  • PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research,
  • the concentration of each set of primer was:- Marker pmol/rxn
  • Each single cell was treated with 5 ⁇ L of 0.624 mg/ml Proteinase K.
  • the single cells were then subjected to the following heating program: a) 50°C for 30 minutes b) 95°C for 15 minutes
  • the cells were then ready for master-mix to be added and subsequent thermal cycling conditions as follows:-. a) 95°C for 1 minutes b) 94°C for 40 seconds c) 57°C for 60 seconds d) 72°C for 40 seconds e) Go to (b), 44 times f) Hold at 4°C.
  • Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJresearch, Geneworks). Master-mix for the PCR was made using 1.2 U of Hot Star Taq (Quiagen) per reaction in all single cell, lx PCR buffer (which contains 1.5mM Magnesium Chloride) were added (provided with the Hot star taq). dNTP's were added to reach 0.2mM concentration. Primers were added as described above. The master-mix was made up to 17.5ul using Milli-Q sterilised water.
  • PBS Phosphate buffered saline
  • Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen). lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24 ⁇ l using Milli-Q sterilised water. The primer concentrations were:-
  • the electropherogram in Figure 11 shows a 9 primer set multiplex from a single amniotic cell that has a 1 in 9 billion chance of two individuals having the same genetic fingerprint.
  • PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's ⁇ were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24 ⁇ l using Milli-Q sterilised water.
  • PCR primer concentrations were:- Marker pmol/rxn
  • FIG. 13 shows a amniotic low copy number sample subjected to 10 primer set multiplex.
  • the PCR amplified genetic markers were AMEL, D13S631, D18S851, DYS14, D18S391, D13S317, D21S11, D13S258, D18S51, D21S1412 PCR cycling parameters were:- a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to b, 39 times f. 72°C for 10 minutes g. Hold at 4°C.
  • An amniotic cell suspension was added at 1.5 ⁇ l (Stored in PBS) and run in a 96 well 200 ⁇ l plate on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units of Amplitaq Gold (Applied Biosystems) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24 ⁇ l using Milli-Q sterilised water.
  • Application of multiple genetic tests according to the invention may be applied, for example, to genetic analysis such as prenatal diagnosis using nucleic acids from isolated fetal cells such as from PAP smears, amniotic fluid or other nucleic acids such as free fetal DNA in maternal blood supply.
  • Figure 14 shows an electropherogram of a 10 primer set multiplex PCR consisting of simultaneous detection of:
  • the DNA was obtained using PCR on a single buccal cell This PCR contains CF1, AMEL, D13S631, D18S851, DYS14, D13S391, D13S317, D21S11, D13S258 & D18S51 Primer concentrations were (per reaction): -
  • PCR cycling parameters were:- a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at 4°C.
  • the single buccal cell was added at 1.5 ⁇ l (Stored in PBS) and run on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using

Abstract

Methods of selection and amplification of genetic markers for genetic testing are provided, and in particular, to facilitate multiplex PCR amplification from limiting amounts of target nucleic acid (i.e. 1 ng, or less, nucleic acid or the amount of nucleic acid contained in 200 or fewer cells). The method of selecting a plurality of genetic markers as targets for nucleic acid amplification includes the step of selecting each of said plurality of genetic markers according to a heterozygosity index, wherein the heterozygosity index is 0.5 or greater. The method of nucleic acid sequence amplification includes a step of using a nucleic acid sequence amplification technique and at least nine primer pairs in combination to amplify a plurality of respective genetic markers from a limiting amount of nucleic acid samples. The methods of the invention are generally applicable to improved genetic diagnostic and screening methods such as prenatal diagnostic testing, fetal sex determination and genetic identification, such as by DNA fingerprinting, in organisms such as bacteria, humans and other animals where available target nucleic acid is limiting. The nucleic acid amplification method of the invention is also applicable to forensic analysis of degraded, old, ancient, difficult and or other low-abundance samples that have hitherto been difficult to amplify and identify.

Description

TITLE MULTIPLE GENETIC MARKER SELECTION AND AMPLIFICATION FIELD OF THE INVENTION THIS INVENTION relates to selection and amplification of genetic markers for genetic analysis. More particularly, this invention relates to selection of genetic markers and primers to facilitate multiplex PCR amplification from limiting amounts of nucleic acid. The methods of the invention are generally applicable to improved genetic testing methods including but limited to diagnostics and screening, for example prenatal diagnostic testing, fetal sex determination and genetic identification, such as by DNA fingerprinting, in organisms including but not limited to bacteria, plants, humans and other animals where available nucleic acid is limiting. The nucleic acid amplification method of the invention is also applicable to by genetic identification of degraded, old, ancient, difficult and or low-abundance samples that have hitherto been difficult to amplify, detect and/or identify. BACKGROUND OF THE INVENTION
Conventional genetic analysis generally consists of a single test on large amounts of sample, for example diagnosis of ΔF508 (major cystic fibrosis mutation) from DNA extracted from a relatively large volume (5 mL) of blood.
Multiple diagnoses by amplification techniques such as multiplex polymerase chain reaction (PCR) have been obtained from large samples such as for forensic DNA fingerprinting, usually requiring a minimum of 200 cells. Or alternatively, single or few diagnoses have been obtained from small samples such as single cells, for example, in pre-implantation genetic diagnosis.
One system that has been used for multiple diagnoses, particularly in prenatal diagnosis, is quantitative PCR using polymorphic short tandem repeats (STRs; Mansfield, 1993, Hum Mol Genet. 2 43-50).
Quantitative PCR accurately determines the amount of PCR product from each allele permitting the ratio of product quantity between alleles to be calculated thus determining aneuploidy status. Even though quantitative PCR was first described in the early 1990s, there are few reports in clinical prenatal diagnosis (Adinolfi et al., 1995.
Bioessays 17 661-664.). A modification of this quantitative PCR technique known as multiplex fluorescent PCR (MF-PCR) has demonstrated the feasibility of using quantitative PCR in clinical prenatal diagnosis even though several thousand cells are still required (Findlay et ah, 1994, Human Reproduction 9 23; Findlay et ah, 1998a, Prenatal Diagnosis 18 1413-1421; Findlay, 1998, Single cell PCR: Theory. Practice and Applications. Clinical Applications In PCR. Edited by Y-M.D. Lo, published by Humana Press; Findlay et ah, 1998b, Molecular Pathology 51 164-168; Findlay et al., 1998c, Journal Assisted Reproduction and Genetics 15 265-274; Findlay et ah, 1998d, Clinical Genetics 53 92-95; Toth et al, 1998, American Journal of Obstetrics 178 1101- 1102; Tόth et ah, 1997, American Journal of Human Genetics 61 807; Verma et al., 1998, Lancet 4;352 (9121) 9-12; Peril et al, 1999a, J. Med. Genet 36 300-3; Peril et al, 1999b, Molecular Human Reproduction 5 1176-9; Schmidt et al, 2000, Mol. Hum. Reprod 6 855-860).
Four main types of STR signal are obtained depending on trisomy status, three of which are shown in Figure 1. Trisomic samples produce either a triallelic signal (three peaks of similar size) seen in Panel 1, or a diallelic or double-dose signal (two peaks, one of which is approximately twice the size of the other) seen in Panel 2. Disomic samples produce a heterozygous signal (double peaks of similar size) seen in Panel 3. Homozygous signal showing a single peak (not shown) are regarded as uninformative, as they can be obtained from both disomic and trisomic samples.
Diallelic signals are partially informative with triallelic signals being the most informative.
Multiplex fluorescent PCR provides two main advantages. Firstly, the multiplex system provides multiple diagnoses, using either linked markers to confirm results or to expand the scope of the test to multiple chromosomes or perform other diagnoses. Secondly, the use of fluorescent markers significantly increases the threshold of detection almost 1000 fold (Findlay et al, 1995, Human Reproduction 10 1005). Generally, however, it has not been possible to use these techniques to diagnose trisomies at or near the single cell level (Findlay, 1998, supra; Findlay et al, 1998a & 1998c, supra; Findlay et al, 1999, Journal of Assisted Reproduction and Genetics 16 199-206). Fluorescent, multiplex PCR has been shown a reliable and accurate method for determining sex (Salido et al, 1992, Am. J Human genetics 50 303; Findlay et al, 1994a, Human Reproduction, 9 23; Findlay et al., 1994b, Advances in Gene Technology: Molecular Biology and Human Genetic Disease. Vol 5, page 62. Findlay et al, 1995, Human Reproduction 10 1005-1013; Findlay et al, 1998c, supra diagnosing genetic diseases such as cystic fibrosis (Findlay et al, 1995, supra), detecting chromosomal aneuploidies and in genetic analyses for genetic identification, such as typically referred to as DNA fingerprinting (Findlay et al, 1997, Nature 389 355-356). The reliability and accuracy rates of fluorescent PCR compare very well with other diagnostic techniques including fluorescent in situ hybridisation (FISH) and Primed In Situ Synthesis (PRTNS; Findlay et al, 1998, J. Assisted Reproduction & Genetics 15 257).
OBJECT OF THE INVENTION The present inventors have sought to improve the quality of information obtainable from amplification of multiple genetic markers in applications relating to genetic testing and identification, such as in prenatal diagnosis, genetic disease screening and testing and DNA fingerprinting, such as in forensics. A problem associated with improving the performance of diagnostic nucleic acid sequence amplification, particularly from limiting amounts of nucleic acid template, is that the more genetic markers are amplified in a multiplex reaction, the more compromised is the quality of the information obtained from the reaction. To solve this problem, the present inventors have improved the selection of genetic markers that can be amplified in combination, the number of genetic markers that can be amplified in combination and the efficiency of amplification from limiting amounts of nucleic acid template
It is therefore an object of the invention to provide improved genetic marker selection.
It also an object of the invention to provide improved multiplex nucleic acid sequence amplification, particularly from limiting amounts of nucleic acid template. SUMMARY OF THE INVENTION In one aspect, the present invention provides a method of selecting a plurality of genetic markers as targets for nucleic acid sequence amplification, said method including the step of selecting each of said plurality of genetic markers according to a heterozygosity index, wherein said heterozygosity index is 0.5 or greater. Preferably, said heterozygosity index is 0.7 or greater.
More preferably, said heterozygosity index is 0.9 or greater.
In another aspect, the invention provides a method of producing one or more primers for amplification of each of a plurality of genetic markers selected according to the first aspect of the invention, said method including the step of selecting a nucleotide sequence for each of said one or more primers so that upon amplification of said genetic marker using said one or more primers, a resultant amplification product has a molecular size in the range 50-3000 bp.
Preferably, the molecular size is in the range 50-1000 bp.
More preferably the molecular size is in the range 80-500 bp. Even more preferably the molecular size is in the range 100-400 bp.
Primers constructed according to this aspect may be degenerate or non- degenerate as is well understood in the art.
In yet another aspect, the invention provides a method of nucleic acid sequence amplification including the step of using a nucleic acid sequence amplification technique and at least nine primer pairs in combination to amplify a plurality of respective genetic markers from a limiting amount of nucleic acid sample.
In particular embodiments, at least ten, eleven, twelve, thirteen, fourteen, fifteen and sixteen primer pairs are used to amplify said respective genetic markers.
Preferably, each said primer pair amplifies a respective genetic marker. Preferably, nucleic acid sequence amplification is performed using PCR.
Preferably, PCR is fluorescent multiplex PCR.
Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. BRIEF DESCRIPTION OF THE FIGURES AND TABLES Reference is now made to non-limiting embodiments of the present invention described by way of example with reference to the accompanying figures and tables. TABLE 1: Examples of genetic markers and primers and, in selected examples, heterozygosity indices (labelled as "heter" for convenience). Expected amplification product sizes may be obtained by consulting databases such as Genbank using the accession number or marker designation listed. The molecular fragment sizes and corresponding primer sequences listed in Table 1 may be readily altered or manipulated by persons skilled in the art. Accordingly, Table 1 provides non-limiting examples of primers and resultant amplification fragment sizes applicable to each genetic marker. A reference to the DYS14 marker is Lo et al, 1993, Hum. Genet. 90 483. Primers are attributed SEQ ID NOS:l-92 in order of appearance in Table 1.
TABLE 2: Non-limiting examples of fluorescently-labeled primers and corresponding genetic markers applicable to multiple genetic diagnoses. TABLE 3: Non-limiting examples of fluorescently-labeled primers and corresponding genetic markers applicable to DNA fingerprinting. TABLE 4: Comparison of FISH, PRTNS and fluorescent multiplex PCR techniques for preimplantation genetic diagnosis (PGD). TABLE 5: Single cell DNA fingerprinting improvements. The new method is that described herein; the published method is that described in Findlay et al, 1997, Nature 389 355-356. !Full profiles provide highest possible specificity. However as more STR markers are added it becomes more likely that one or more will fail or be compromised. Although higher full-profile values are generally better, the inclusion of additional markers with higher specificity more than compensates; for example add four markers but lose one = net gain of three. The "acceptable profile" category is much more important and significant. 2Surplus alleles. Additional alleles in conjunction with true alleles. Causes a marker to be defined as uninformative. Lower percentages are therefore better. 3False alleles. Additional alleles in place of true allele. Extra-allelic peaks caused by contamination, somatic mutation or PCR generated non-allelic peak artefacts. False alleles may result in misdiagnosis and must therefore be minimized as much as possible. In this improved system, such false alleles occur rarely (<1%). More than 2 additional peaks in a profile or from 18 negatives were not observed. When false alleles are observed the marker, but not the profile, is defined as uninformative. Lower values are therefore better. 4Allele dropout results in reduction of specificity. Lower values are again better. However as more STR markers are added it becomes more likely that one or more individual alleles will fail. Again the inclusion of additional markers more than compensates. For example add four markers but lose one = net gain of three.
TABLE 6a and 6b: Example demonstrating twin heterozygosity. Table 6a shows allele sizes obtained for each marker. It can be seen that the genetic identification allele sizes for twin 1 are identical to that of twin 2 thus indicating that the twins are identical twins. Table 6b demonstrates maternal or paternal derivation of each allele thus indicating maternity and paternity.
TABLE 7: Example demonstrating twin heterozygosity. Table 7 shows allele sizes obtained for each marker. It can be seen that the genetic identification allele sizes for twin 1 are different to that of twin 2 thus indicating that the twins are not identical twins.
FIG. 1 : Quantitative fluorescent PCR in prenatal diagnosis. Panel two shows 2: 1 ratio or diallelic signal.
FIG. 2: Multiplex fluorescent PCR from a single cell sample with nine out of nine genetic markers present.
FIG. 3: Limited nucleic acid template sample subjected to eleven (11) primer set multiplex PCR. In this example 10 of 11 markers were amplified.
FIG. 4: Eleven (11) primer set multiplex PCR on single diploid cell. 11 of 11 markers were amplified FIG. 5: Eleven (11) primer set multiplex PCR on single sperm (haploid cell).
Note all markers are homozygous, which is indicative of haploid cells. 11 of 11 markers were amplified
FIG. 6: Sixteen (16) primer multiplex PCR on single diploid cell. 16 of 16 markers amplified FIG. 7: Genetic identification of single fetal cell isolated from PAP smears using nine (9) primer pairs. 9 of 9 markers amplified. Maternal genetic identification is also shown to demonstrate that both fetal signal and maternal signals share common alleles (indicating maternity), but the fetal cell has inherited other alleles from a paternal source, consistent with Mendelian inheritance.
FIG. 8: Genetic identification of single fetal cell isolated from PAP smear using eleven (11) primer pairs.
FIG. 9: Nine (9) primer pair multiplex PCR demonstrating twin heterozygosity using limited amount of amniotic fluid. Results indicate both twins identical. FIG. 10: Nine (9) primer pair multiplex PCR demonstrating twin heterozygosity using limited amount of amniotic fluid. Results indicate both twins non-identical. FIG. 11 : Multiplex fluorescent PCR from a single cell sample with nine (9) out of nine (9) genetic markers present.
FIG. 12: DNA fingerprint obtained from hairshaft using eleven (11) primer fluorescent multiplex PCR.
FIG. 13: Ten (10) primer pair fluorescent multiplex PCR demonstrating genetic diagnosis of trisomy status from limited amount of amniotic fluid.
FIG. 14: Ten (10) primer pair multiplex demonstrating simultaneous diagnosis of single-gene defect (cystic fibrosis), sex, trisomy status and genetic identification. DETAILED DESCRIPTION OF THE INVENTION The invention described herein relates to nucleic acid sequence amplification of multiple genetic markers, and methods of selecting genetic markers to improve the efficiency of marker amplification.
With regard to genetic marker selection, the present invention is based on the realization that there are two main considerations when selecting a genetic marker for nucleic acid sequence amplification. • Heterozygosity. Heterozygosity is defined as the presence of different alleles of a gene at one or more loci. Heterozygosity occurs when a diploid organism or cell has inherited different alleles at a particular locus from each parent. Heterozygosity index is a measure of the likelihood of marker alleles being different within individuals i.e. having two alleles rather than one. For example alleles from markers with low heterozygosity are more likely to be identical or be homozygous within an individual or population. Markers with higher heterozygosities are more likely to provide triallelic (most informative) results (see Figure 1), if the sample is trisomic. It is therefore necessary to choose markers with as high heterozygosity (dp) values as possible. • Fragment size. The optimal fragment size window is between 100-400bp although 80bp to 500bp, 50bp to lOOObp or even 50 bp to 3000 bp can be used. Fluorescent systems for fragment detection have increasingly limited detection when fragment size is less than 80bp due to interference from primer dimer. Fragment sizes that are large, (e.g. greater than 500bp), even though they may not accurately be sized can still be used to identify multiple peaks and triallelic results. In general the larger the fragment size the more time it takes for results to be obtained. As most diagnostic laboratories require results as quickly as possible, smaller fragments would therefore be most preferred. Additional considerations include:-
• Appropriate chromosome. It is necessary to choose a marker that will accurately reflect the test being performed. For example, if one is attempting to determine the number of copies of chromosome 21, a marker on chromosome 21 is most likely to be the most appropriate. • Fluorescent labeling. Using fluorescent labeled primers to combine markers in a multiplex with markers of similar or overlapping fragment ranges. The choice of fluorescent label is very important since marker allele sets can overlap with each other. Overlap with another marker would make the marker of limited value since each marker may then be indistinguishable from the other. For example if one marker was heterozygous and the other homozygous this would show as a triallelic response incorrectly indicating a trisomy. When marker size sets do overlap, the marker could be labelled with a differently coloured fluorochrome thus allowing identification of each marker. The present invention therefore provides a substantial improvement in the efficiency of genetic marker selection such as for the purposes of selecting STRs that allow PCR amplification of multiple genetic markers for applications including genetic identification (for example, human embryo identification and forensics), genetic diagnosis and screening (for example, pre-implantation genetic diagnosis after IVF and from fetal cells obtained from cervical smears, CVS or amniocentesis), although without limitation thereto.
Furthermore, the present invention provides multiplex PCR amplification using at least nine primer pairs to amplify discrete genetic markers from limiting amounts of nucleic acid sample in a highly efficient manner. For example, nine (9) informative genetic markers were successfully amplified in 69 of 69 multiplex amplifications using nine (9) primer pairs and nucleic acid samples from single buccal cells. The present invention also contemplates amplification of up to and in excess of sixteen (16) genetic markers as will be described in more detail hereinafter.
It will be apparent to the skilled person that the present invention is broadly applicable to "genetic analysis". As used herein, genetic analysis and genetic diagnosis are used interchangeably and broadly cover detection, analysis, identification and/or characterization of genetic material and includes and encompasses terms such as, but not limited to, genetic identification, genetic diagnosis, genetic screening, genotyping and DNA fingerprinting which are variously used throughout this specification.
For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native or recombinant form. The term "nucleic acid" as used herein designates single-or double-stranded mRNA, RNA, cRNA, RNAi and DNA, said DNA inclusive of cDNA and genomic DNA.
A "polynucleotide" is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has less than eighty (80) contiguous nucleotides. By "genetic marker" or "marker''' is meant any locus or region of a genome. The genetic marker may be a coding or non-coding region of a genome. For example, genetic markers may be coding regions of genes, non-coding regions of genes such as introns or promoters, or intervening sequences between genes such as those that include tandem repeat sequences, for example satellites, microsatellites, short tandem repeats (STRs) and minisatellites, although without limitation thereto.
A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example. Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999); strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J. Am. Chem. Soc. 118 1587 and International application WO 92/01813 and by Lizardi et al, in International Application WO 97/19193; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17 1077; and Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395.
The abovementioned are examples of nucleic acid sequence amplification techniques but are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein. As used herein, "multiplex amplification" or "multiplex PCR" refers to amplification of a plurality of genetic markers in a single amplification reaction.
In particular embodiments, the invention provides "fluorescent PCR". This system uses fluorescent primers and an automated analyser such as a DNA sequencer to detect PCR product (Tracy & Mulcahy, 1991, Biotechniques 11 68-75). Fluorescent PCR has improved both the accuracy and sensitivity of PCR for genotyping (Ziegle et al, 1992, Genomics, 14 1026-1031; Kimpton et al, 1993, PCR Methods and Applications 3 13-22).
In particular embodiments fluorescent amplification products are electrophoresed using gel or capillary systems and pass a scanning laser beam, which induces the tagged amplification product to fluoresce. The DNA sequencer combined with appropriate software is generally known as a "Genescanner". Stored data can then be analysed to provide product sizes and the relative amount of amplification product in each sample.
A preferred nucleic acid sequence amplification technique is PCR. As used herein, an "amplification product' refers to a nucleic acid product generated by a nucleic acid amplification technique.
A "primer" is usually a single-stranded oligonucleotide, preferably having 12-
50 contiguous nucleotides which, for example, is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent
DNA polymerase or Sequenase™.
Non-limiting examples of primers that may be used in combination are primers capable of amplifying genetic markers (STR loci) index listed in Table 1 .
For fluorescent PCR, each said primer may be fluorescently-labeled to produce a fluorescently-labeled primer pair. Fluorescent labels are well known in the art and include but are not limited to TET, FAM, HEX as for example described in Table 2.
Other fluorescent labels potentially useful according to the invention include but are not limited to CyDyes™ such as Cy2, Cy3, C3.5, and Cy5.
Non-limiting examples of fluorescent-labeled primers are set forth in Table 2. Primer synthesis and incorporation of fluorescent labels are well known in the art and labeled, synthetic primers are readily available from commercial sources. However, an example of primer synthesis methodology is provided in Chapter 2..11 of Current Protocols in Molecular Biology Ausubel et al. Eds (John Wiley & Sons, NY, 1996-2001). It will be appreciated by the skilled that the present invention is particularly suited to selection of genetic markers and corresponding primers for the purposes of nucleic acid sequence amplification from limiting amounts of nucleic acid.
As used herein, a "limiting amount of nucleic acid" is an amount of nucleic acid used in a nucleic acid sequence amplification reaction less than 1 ng, preferably less than 500 pg, more preferably less than 200 pg, even more preferably less than 50 pg and in particular embodiments, about 3-6 pg.
It should also be appreciated that a limiting amount of nucleic acid may also relate to the number of cells containing the nucleic acid sample used for amplification. Preferably, the limiting amount of target nucleic acid is obtained from less than
200 cells, more preferably from less than 100 cells, more preferably from less than 50 cells and even more preferably from less than 20 cells.
In particularly preferred embodiments, the limiting amount of nucleic acid is isolated from no more than ten cells, or from a single cell. For example, the present inventors have performed multiplex PCR amplification from a single, haploid sperm cell which comprises about 3-6 pg of DNA.
Although the invention also contemplates use of nucleic acid other than DNA, preferably the nucleic acid is DNA.
More preferably, the nucleic acid is genomic DNA. Suitable sources of cells from which DNA may be obtained include, but are not limited to, buccal cells, sperm cells, hair follicle cells, skin cells, epithelial cells, nucleated cells circulating in blood, embryonic cells, fetal cells such as obtained from fetal blood, CVS or amniocentesis samples or cervical (PAP) smears, corneal cells, cell or tissue biopsies, or any other cells or tissues from which genetic material can be obtained.
In one embodiment, the invention is applicable to genetic analysis from small numbers of cells such as for the purposes of prenatal diagnostic testing or screening, fetal sex determination and genetic identification by DNA fingerprinting.
Cellular sources such as described above may be of any organism inclusive of plants, bacteria and animals.
Preferred sources of nucleic acids are mammals, preferably humans. The invention also contemplates genetic analysis of non-human samples such as from cows, sheep, horses, pigs and the like, although without limitation thereto.
Also contemplated are other vertebrate sources of nucleic acid such as birds. Non-limiting examples are domestic birds such as chickens, ducks and geese. The skilled person will also appreciate that nucleic acids may be obtained from non-cellular sources such as viruses.
It will also be understood that nucleic acids are not necessarily directly obtained from their cellular or non-cellular source organism. As will be apparent from this specification, nucleic acids may be obtained from objects such as paper, documents, clothing, bedding, motor vehicles, physical fingerprints, weapons, furniture and building fixtures and a variety of substrates such as biological fluids and ink although without limitation thereto, such as for the purposes of genetic identification.
For the purposes of obtaining nucleic acid samples for amplification, cells or nucleic acid material may or may not require isolation by techniques as are well known in the art. These include but are not limited to densitometric separation such as by gradient centrifugation through media such as Metrizamide™, Ficoll™ and Percoll™, biochips, micro-manipulation, pulse field separation, differential lysis and antibody- mediated isolation such as by panning, magnetic bead separation or fiow-cytometric (FACS) sorting. An example of densitometric cell separation is provided in Albright et al, 1986,
Cytometry 7 536, where the use of centrifugal separation of cells in sputum specimens is described.
Isolation of antibody-labeled cells by magnetic bead separation is well known in the art for which kits are commercially available (for example the Dynal MPC cell separation kit).
With regard to FACS sorting, generally applicable flow cytometry methods are described in Practical Flow Cytometry (Second Edition) by Howard M. Shapiro and in Chapter 5 of Current Protocols in Immunology, Coligan et al. Eds (John Wiley & Sons NY, 1995-2001 Prenatal diagnosis using nucleic acids isolated from fetal cells
In a particular embodiment, the invention provides a method of prenatal genetic diagnosis of a fetus wherein a nucleic acid sample for amplification is obtained from one or more fetal cells isolated from a pregnant individual (i.e the mother). As used herein, the word "fetal" includes embryonic cells at any developmental stage from any species.
Fetal cells may be isolated by any cell isolation method.
Said one or more fetal cells may be isolated from any pregnant mammal.
Preferably, said one or more fetal cells are isolated from a pregnant human. When a fetus is at increased risk for genetic defects such as chromosomal anomalies, typically prenatal diagnosis is by invasive procedures such as either chorionic villus sampling (CVS) in the late 1st trimester or amniocentesis in the 2nd trimester of pregnancy. By the third trimester, a combination of CVS and amniocentesis, or even fetal blood sampling, may be necessary. A rapid, less-invasive and low cost method of prenatal diagnosis involves genetic diagnosis from fetal cells shed into the cervical sump at 6-20 weeks of gestation. These samples are obtained from the cervix by cytobrush in a manner similar to a PAP smear which is similar to but less invasive than invasive transcervical sampling. Although promising, there are four major difficulties. Firstly the need to obtain the large numbers of fetal cells normally required for genetic analysis. Secondly the isolation of fetal cells from the cervical sample is extremely difficult as recent results suggest that fetal cells could be isolated and diagnosed in only -22% of cases due to the presence of "contaminating" maternal cells. Previous approaches have generally concentrated on isolating fetal cells by morphology or cell sorting. Unfortunately, morphology grading is extremely time-consuming, expensive and generally unreliable and inaccurate. Alternative cell sorting techniques involve antibody-labelled slides to capture fetal cells, which is generally unspecific and can result in major maternal contamination and misdiagnosis or insufficient fetal cells. Thirdly the difficulty of obtaining genetic diagnosis from small cell numbers. Although fetal cells have been identified in cervical samples (mainly by identifying male cells within the sample) aneuploidy screening (the primary reason for prenatal diagnosis) cannot usually be performed nor diagnosis made if the fetus is female. Finally, although sampling is theoretically much safer than CVS and amniocentesis as PAP smears have been taken during pregnancy for many years, relative safety remains to be fully evaluated. According to this embodiment of the present invention, it is preferred that said fetal cells are present in a maternal cavity, such as the uterus, or endocervical canal sample, particularly a transcervical sample. Methods of isolating fetal cells include but are not limited to cervical cotton swab, cytobrush, aspiration of cervical mucus, lavage of the endocervical canal and uterine lavage. Samples can be obtained from transcervical aspiration of mucus from just above the internal os or the lower uterine cavity. Another isolation method which may be used is lavage which is generally conducted with a saline wash, but other isotonic solutions are suitable. Typically, endocervical lavage with 5- 10ml or intrauterine lavage with 10-20 ml saline provides sufficient fetal cells upon separation from maternal cells. The sample may be collected using a combination of methods.
Preferably, cell samples are isolated from a female human in the first trimester of pregnancy or when the fetus is between 6 to 20 weeks gestation.
Preferably, to aid fetal cell separation, clumps of cells are preferably treated to obtain a suspension of single cells. The clumps may be separated by techniques known to a skilled person, such as enzymatic, chemical or mechanical separation. For example, enzymatic separation may utilise protease or trypsin. Chemical separation may utilise acetyl cysteine and mechanical separation may involve gentle teasing, aspiration or micromanipulation.
The number of fetal cells in the sample varies depending on factors including the age of the fetus, method of sampling, number and frequency of samplings, the volume of washing in each lavage where lavage is used and the volume aspirated.
Maternal uterine cavity or endocervical canal samples typically contain at least two main types of nucleated fetal cells: cytotrophoblasts and syncytiotrophoblasts cells. Fetal cells can be isolated either by selecting fetal cells from maternal cells (positive selection) or isolating the maternal cells from the fetal cells (negative selection) or most preferably a combination of both. Preferably, the nucleated fetal cells are retained in the purified sample.
When 100% purity is desired, one method to isolate fetal cells is micromanipulation. In another method, for example, cell suspensions containing an individual cell per a preselected volume of suspension medium can be prepared by limiting dilution. Drops containing individual cells can placed in suitable container (e.g. 96 well plates) and examined visually with a fluorescent microscope to identify single- labelled (or unlabelled) cells.
For PCR analysis, analysis can be performed using a single, identified fetal cell. Alternatively, ways can be envisaged of identifying monozygosity (indicative of the presence of a monogenic disease) in a mixed cell population containing minimal fetal material including as few as one fetal cell in 100 cells. Following sorting, the separated cells can be washed twice in a physiologic buffer and resuspended in an appropriate medium for any subsequent analysis to be performed on the cells. Following recovery or isolation methods, the fetal cells can be used in the same manner as fetal cells obtained by other methods such as amniocentesis and chorionic villus biopsy. The cells can be used as a source of DNA for analysis of the fetal alleles, as by polymerase chain amplification for example. PCR analysis methods may be used to detect, for example, fetal sex, beta thalassemia, phenylketonuria (PKU), and Duchennes muscular dystrophy without limitation thereto.
Alternatively, the cells can be cultured in a similar manner as material biopsied for karyotyping analyses. However, the incubation period may be significantly shortened if a DNA content of greater than or equal to 2C is used as a selection criterion. In another embodiment, the present invention provides a method of prenatal analysis using nucleic acids isolated from fetal cells isolated by but not limited to invasive procedures such as from fetal blood, amniocentesis or CVS.
Although widespread, conventional cytogenetic diagnoses from amniotic fluid have significant disadvantages including high cost and significant delay (~2 weeks) which results in a lengthy wait for reassurance for a healthy pregnancy or the prospect of a late termination for the parents. Alternative sampling methods such as CVS or diagnostic techniques such as FISH (Fluorescent in situ hybridisation) can provide more rapid results. However CVS has higher miscarriage risk; requires higher technical skill and requires physical referral to specialised tertiary centres with additional inconvenience, stress, and costs to families and Health Care. FISH has interpretation difficulties and higher failure rate when samples are small; high cost; limited throughput and is labour intensive (~10 samples per day) and has limited analysis potential (~5 of the common chromosomes defects).
An alternative diagnostic method is quantitative PCR using polymorphic short tandem repeats (STRs) to accurately determine PCR product ratio from each allele and thus aneuploidy status. However the high numbers of cells required and profile interpretation difficulties result in few clinical applications.
Multiplex fluorescent PCR is a viable alternative for clinical prenatal diagnosis. MF-PCR techniques are becoming adopted overseas in both public and private laboratories.
However these techniques have had several limitations:
1. Limited marker sets with limited diagnostic capability which, for example, generally only determine aneuploidy and/or sex.
2. Large amount of samples required as DNA extraction is required. 3. Samples are not genetically identified as being fetal thus having. potential for misdiagnosis from contamination.
4. Additional cost and time for DNA extraction.
5. Low range of diagnoses available.
The present invention in one embodiment provides improved multiplex nucleic acid sequence amplification on limited samples to overcome these difficulties. This invention in one embodiment will significantly improve diagnostic confidence, capability as well as reduce cost and time.
Preferably, cell samples are isolated from a female human in the first trimester of pregnancy or when the fetus is between 6 to 20 weeks gestation and may consist of amniotic fluid or samples from the chorionic villi. The number of fetal cells in the sample varies depending on factors including the age of the fetus, method of sampling, skill of operator, number and frequency of samplings, the amount of sample obtained in each procedure and the volume aspirated.
Amplification methods such as PCR analysis may be used to detect, for example, fetal sex, beta thalassemia, phenylketonuria (PKU), and Duchennes muscular dystrophy without limitation thereto.
Forensic samples
In another embodiment, the present invention relates to genetic analysis or genetic identification by "DNA profiling" or commonly known as DNA fingerprinting of samples. In this regard, cellular and/or non-cellular nucleic acid samples can be obtained from a variety of sources including but not limited to forensic samples (such as clothing, bedding, motor vehicles, physical fingerprints, weapons, furniture and building fixtures and a variety of substrates such as biological fluids and ink), documents or other substrates such as ink or paper and ink derived therefrom, archaeological or other old or ancient samples, samples obtained for the purposes of personal identification, biological samples or clinical samples such as used for genetic identification, testing, screening and/or diagnosis of genetic diseases, sexing and detection of chromosomal abnormalities.
DNA profiling is an extremely powerful method for forensic identification with current prior art achieving power of discrimination in excess of 1 in 10 billion.
However, many forensic PCR methods require many hundreds of cells to maintain the necessary high rates of reliability and accuracy. Forensics identification has been attempted on smaller samples with limited success. For example, STR profiling systems have been applied to low cell samples such as cigarette butts (Torre and Gino, 1996, J Forensic Sci. 41 658-9) and from cells left on pens, car keys, etc (van
Oorschot and Jones, 1997, Nature 387 767). However, these systems either still require similarly large amounts of DNA for high reliability, or as cell numbers decrease have fewer STR markers therefore markedly decreased discriminating power and reliability.
A particular problem applies in rape cases, particularly multiple rape where semen from multiple sources are present. Conventional forensic analysis requires a clean uncontaminated sample to obtain a DNA fingerprint but, as the semen may be a mixed sample (for example, from each assailant, or assailant and male partner), definitive DNA fingerprints are usually not possible. This leads to a failed forensic test, with the result that there may be insufficient evidence for the prosecution or defence.
The present invention provides an improved method whereby genetic identification by DNA fingerprinting can now be obtained from small samples and or single cells such as single sperm to determine their origin and thus identify each assailant.
In another embodiment, single cells may be obtained from samples which have too few cells for conventional profiling; from samples contaminated by blood or other cell types; from archived cases; old previously solved or unsolved cases; and from physical fingerprints. The single cell DNA fingerprint test described here could be applied for genetic identification to a wide variety of samples and sample types including but not limited to smudged physical fingerprints, single flakes of dandruff, as well as small samples left on weapons, vehicles and other objects. Preimplantation Genetic Diagnosis
IVF success rates have remained relatively constant at only ~ 10-20% per embryo transferred. This may be because a sizeable number of human embryos are chromosomally or otherwise abnormal and therefore unable to implant, or form or maintain a pregnancy. It has been possible since 1990 to diagnose genetic defects from single embryonic cells removed from embryos (preimplantation genetic diagnosis (PGD) also alternatively known as preimplantation diagnosis (PID)). There are three main applications for PGD: sex, single gene defects and aneuploidy e.g. trisomy diagnosis. In general FISH (fluorescent in situ hybridisation) is used for sex or aneuploidies and PCR for single gene defects.
Single cell fluorescent PCR has previously shown to be highly reliable (97%), highly accurate (97%), rapid (6hrs) and wide ranging (simultaneous diagnoses of sex, single gene defects and trisomies) (Findlay et al, 1995, Human Reproduction 10 1609- 1618). However such testing has been limited to a limited number (upto and including 8) of markers, which limits use. Single cell fluorescent PCR can also determine a DNA fingerprint from a single cell therefore minimising the risk of misdiagnosis due to contamination (Findlay et al, 1995, Human Reproduction 10 1005-1013; Findlay, 1996, Human Reproduction Update 2 137-152; Findlay et al, 1997, Nature 389 355-356; Henderson et al, 2001, Cornea 20 400-403). Again such testing has been limited to a limited number (up to and including 8) of markers, which severely limits use in genetic identification particularly since a major source of contamination is parental DNA which share common alleles with the embryonic cell thus significantly decreasing specificity of discrimination of the DNA fingerprint. Fluorescent PCR can be favourably compared to other techniques as shown in Table 4.
DNA fingerprinting of embryonic cells allows individual embryos to can be genetically "tracked" from the 6-8-cell stage to birth and beyond. This makes it possible to determine which pregnancy resulted from which embryo. The present invention allows DNA fingerprinting to be performed on single cells with a specificity greater than 10 billion to 1, far in excess of any other single cell genotyping system and far in excess of prior art (Findlay et al, 1997, Nature 389 355-356) single cell DNA fingerprinting at -100 million to 1. In the case of IVF, this embodiment of the present invention provides but is not limited to: a clinical tool that provides a quality control mechanism; patient reassurance that correct embryos are identified for transfer; determination of separate pregnancy rates in multiple embryo transfer e.g. when different treatments such as ICSI and non-ICSI embryos are transferred together; minimization of contamination rate in pre-implantation genetic diagnosis using PCR; as a research tool for example in comparing embryonic phenotype with genotype; and can be used to determine heterozygosity of twins in multiple births. Furthermore, the method of the invention may be used to provide accurate and absolute correlation of embryo quality with pregnancy and might be used to accurately compare differing culture conditions. For example embryos cultured in two different media can be transferred to the same woman and an accurate pregnancy rate per media derived. Patient reassurance is also improved by the PCR method of the invention by confirming that embryos transferred are genetically derived from parents. It should also be appreciated by the skilled that the invention may be used for genetic analysis such as PGD or prenatal diagnosis or screening from non-human sources. Such non-limiting examples include PGD or genetic screening of an increased number of a wide variety of genetic traits to improve qualities from domestic animals such as cattle.
It should also be appreciated by the skilled that the invention may be used for a wide variety or purposes and applications where samples are limited in amount or availability and/or where maximal information from genetic testing is required or useful. So that the invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.
EXAMPLES Heterozygosity Index The eukaryotic genome is densely populated with islands of short sequences that are repeated over and over in small to large arrays called minisatellites and microsatellites. Another term commonly used to describe these sequences is variable number tandem repeats or VNTRs.
For a given repetitive locus, the number of repeats is highly variable among individuals and heterozygosity is high (i.e. the number of repeats at the locus is usually different on the two pairs of chromosomes of one individual). Analysing the number of repeats at one or more such loci provides a highly sensitive measure of individual identity and is the preferred technique for forensic DNA typing as means of genetic identification.
Tandem repetitive sequences are classified into three major groups: 1. Satellites are very highly repetitive with repeat lengths of one to several thousand base pairs. These sequences typically are organized as large (up to 100 million bp) clusters in the heterochromatic regions of chromosomes, near centrosomes and telomeres; these are also found abundantly on the Y chromosome. 2. Minisatellites are moderately repetitive, tandemly repeated arrays of moderately-sized (9 to 100 bp, but usually about 15 bp) repeats, generally involving mean array lengths of 0.5 to 30 kb. They are found in euchromatic regions of the genome of vertebrates, fungi and plants and are highly variable in array size. 3. Microsatellites are moderately repetitive, and composed of arrays of short (2-6 bp) repeats found in vertebrate, insect and plant genomes. The human genome contains at least 30,000 microsatellite loci located in euchromatin. Copy numbers are characteristically variable within a population, typically with mean array sizes on the order of 10 to 100.
In general, satellite DNAs show exceptional variability among individuals, particularly with regard to the number of repeats at a given locus. Microsatellite loci are highly polymorphic sequences elements in the human genome, and delineating the repeat lengths of these loci is the basis of most DNA typing systems used in forensic medicine.
Heterozygosity is defined as the presence of different alleles of a gene at one or more loci. Heterozygosity occurs when a diploid organism or cell has inherited different alleles at a particular locus from each parent. Both cases result in mixtures of DNA sequences that have important applications in fields such as forensics, pathology, genetic diagnosis, and evolutionary genetics.
Genetic marker and primer selection Polymorphism in a population is due to the existence of different genetic variants. The basis of variation is thus the number of polymorphic loci together with the number of alleles and their frequency distributions in a population. Based on this concept, markers in Table 1 were checked for the number of different alleles and genetic diversity, both by determining allele frequencies and from data provided publicly through public databases such as GenBank. Markers with higher heterozygosity rates (highly variable) are selected in preference.
PCR protocol for sexing, chromosome 21, 18 and 13 detection A limited number of cells were isolated from amniotic fluid. A mastermix containing the reagents required for the PCR is made up under aseptic conditions. The mastermix contains enough reagents for a number of 25 μl reactions. The primers together with an indication of fluorescent labels for each primer are shown in Table 2, and the composition of the mastermix, per reaction, is as follows:- Reagents Amounτ μl
• lOx PCR buffer 2.5 • MgCl2 1.5 (1.5mM concentration)
• dNTP's 4.0 (1.25mM concentration)
• Taq 0.24 (5Uper μl)
• Distilled waterto make up to 24μl
The mastermix is mixed thoroughly and added to template, or if using a plate, the mastermix is aliquoted and template added to it. The tubes/plate was placed on a thermal cycler and subjected to the following PCR program:-
1. 95°C for 15 minutes
2. 94°C for 30 seconds
3. 59°C for 45 seconds 4. 72°C for 60 seconds
5. Goto step 2, 39 times
6. 72°C for 10 minutes
7. 4°C hold
8. End The PCR uses no oil overlay, as the heated lid of the PCR is sufficient. The
PCR is taken off the block and stored at 4°C until required for electrophoresis. DNA fingerprinting of single/small numbers of cells Cell lysis protocol
1. After cell isolation, single or small numbers of cells are stored at -80°C until needed.
2. Lysis is carried out by adding lμl of Lysis Buffer (200mM KOH, 50mM DTT) to the cell or cells.
3. The mixture containing the cell or cells is spun down with the buffer and heated to 65°C for 10 minutes. 4. 1 μl of Neutralising Buffer (300mM KC1, 900mM Tris-HCl pH 8.3, 200mM HC1) is added to the cell or cells.
5. The cell mixture is spun down and is ready for PCR or stored at -80°C until needed. PCR protocol
A mastermix containing the reagents required for the PCR is made up under aseptic conditions. The mastermix contains enough reagents for a number of 25 μl reactions. The primers together with an indication of fluorescent labels for each primer are shown in Table 3 and the composition of the mastermix, per reaction, is as follows:- Reagents Amount/μl
• lOx PCR buffer 2.5
• MgCl2 1.5 (1.5mM concentration)
• dNTP's 4.0 (1.25mM concentration)
• Taq 0.24 (5Uper μl) • Distilled waterto make up to 24μl
The mastermix is mixed thoroughly and added to lμl of template, or if using a plate, the mastermix is aliquoted and the template is added to it.
The tubes/plate is placed on a thermal cycler and subjected to the following program:- 1. 95°C for 14 minutes
2. 94°C for 60 seconds
3. 57°C for 60 seconds
4. 72°C for 60 seconds
5. Goto step 2, 44 times 6. 72°C for 10 minutes
7. 4°C for ever
8. End
The PCR uses no oil overlay, as the heated lid of the PCR is sufficient. The PCR is taken off the block and stored at 4°C until required for electrophoresis. Multiplex PCR from a single cell sample with nine out of nine genetic markers present Single cells were isolated by micro-manipulation from buccal cell samples Genetic markers are AMEL (1), D13S631 (2), D13S258 (3), D18S851 (4), D18S391 (5), DYS14 (6), D21S11 (7), D21S1411 (8) & D21S1412 (9) as shown in Figure 2.
Primer concentrations were: - AMEL 3.5 pmole
D13S631 9 pmole
D13S258 6 pmole
D18S851 5 pmole
D18S391 10 pmole DYS14 4 pmole
D21S11 7 pmole
D21S1411 10 pmole
D21S1412 13 pmole
PCR cycling programme for the PCR in Figure 2 was: a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at 4°C. The following PCR conditions are used for all single-cell and low copy analysis unless stated differently.
Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix consisted of 2 units taq polymerase (Amplitaq Gold (Applied Biosystems)); lx PCR buffer and 1.5mM Magnesium Chloride (provided with the Amplitaq Gold); dNTP's (0.2mM concentration) and fluorescent and non- fluorescent primers at varying concentrations. The master-mix was made up to 24μl using MiUi-Q sterilised water. Single cells were isolated using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - lμl of PBS drawn with the single cell. DNA analysis was performed using DNA sequencers such as ABI 377 or Megabace 1000 using standard protocols.
Low copy number sample subjected to 10 primer set multiplex PCR Figure 3 shows a low copy number sample subjected to 10 primer set multiplex. Single cells were isolated by micro-manipulation from buccal cell samples. The PCR amplified genetic markers are AMEL (1), D13S631 (2), D18S851 (3), DYS14 (4), D18S391 (5), D13S317 (6), D21S11 (7), D13S258 (8), D18S51 (9), D21S1412 (10). In this example 10 of 10 markers amplified successfully. PCR cycling programme for PCR for Figure 3:- a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at4°C.
Primer concentrations were: -
AMEL 1.4 pmole
D13S631 6 pmole D18S851 5 pmole
D13S258 6 pmole
D18S391 11 pmole
DYS14 4 pmole
D21S11 8 pmole D18S51 3 pmole
D21S1412 12 pmole
D13S317 8 pmole
11 primer set multiplex PCR on single diploid cell
Figure 4 shows an electrophorogram of eleven genetic markers AMEL, THO, D21S11, D18S51, VWA, FGA, D3S1358, D5S818, D7S820, CSF and TPOX amplified from DNA template obtained from a single cell isolated from a buccal cell sample. In this example 11 of 11 markers amplified successfully. PCR cycling parameters were:- 1) 95°C for 10 minutes 2) 94°C for 60 seconds
3) 57°C for 60 seconds
4) 72°C for 60 seconds
5) Go to 2, 44 times
6) 72°C for 10 minutes 7) Hold at 4°C
Single cell samples are added to lul of lysis buffer (200mM KOH/50mM DTT), heated to 65°C for 10 minutes, lul of neutralising buffer (300mM KCl/900mM Tris- HC1, ph8.3/200mM HC1) was then added.
PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride was added. dNTP's were added to reach 0.2mM concentration. Primers were added as described for each individual case. The master- mix was made up to 24μl using Milli-Q sterilised water. Single cells were picked using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - lμl of PBS drawn with the single cell.
PCR primer concentrations:-
Marker pmol/rxn
AMEL 3.5
THO 6.0
D21S11 16.0
D18S51 16.0
VWA 30.0
FGA 6.0
D3S1358 10.0 D5S818 8.0
D7S820 10.0
CSF 8.0
TPOX 3.0 12 primer set multiplex PCR on single sperm (haploid cell)
The electropherogram shown in Figure 5 shows the results of multiplex PCR amplification from DNA template obtained from a single sperm cell. In this example 12 of 12 markers amplified successfully. Primer concentrations were:- Marker pmol/rxn
AMEL(l) 3.5
THO(2) 6.0
D21S11 (3) 16.0
D18S51(4) 6.5 VWA(5) 3.6
FGA(6) 8.0
D3S1358 (7) 6.0
D5S818 (8) 10.0
D7S820 (9) 10.0 CSF (10) 6.0
TPOX (11) 6.0
D13S317 (12) 6.0
The single cells were subjected to lysis prior to PCR. Each single cell had 5μl of
0.624 mg/ml Proteinase K. The single cells were then subjected to the following heating program
1) 50°C for 30 minutes
2) 95°C for 15 minutes
The cells were then ready for master-mix to be added and subsequent following PCR program. 1) 95°C for 1 minutes
2) 94°C for 40 seconds 3) 57°C for 60 seconds
4) 72°C for 40 seconds
5) Go to 2, 44 times
6) Hold at 4°C Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.8U of Hot Star Taq (Quigen) per reaction in all single cell, lx PCR buffer (which contains 1.5mM Magnesium Chloride) were added (provided with the Hot start taq). dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 17.5μl using Milli-Q sterilised water. Single cells were picked using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - lμl of PBS drawn with the single cell.
16 primer multiplex PCR on single diploid cell (16 simultaneous marker sets) Figure 6 shows an electropherogram that demonstrates successful amplification of sixteen (16) genetic markers. In this example 16 of 16 markers amplified successfully. Single cells obtained from buccal cell samples were subjected to lysis prior to PCR. Each single cell had 5μl of 0.624 mg/ml Proteinase K. The single cells were then subjected to the following heating program: a) 50°C for 30 minutes b) 95°C for 15 minutes
The cells were then ready for master-mix to be added and subsequent following PCR program. a) 95°C for 1 minutes b) 94°C for 40 seconds c) 57°C for 60 seconds d) 72°C for 40 seconds e) Go to (b), 44 times f) Hold at 4°C Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.8U of Hot Star Taq
(Quigen) per reaction in all single cell, lx PCR buffer (which contains 1.5mM
Magnesium Chloride) were added (provided with the Hot star taq). dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 17.5μl using Milli-Q sterilised water. Single cells were picked using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - lμl of PBS drawn with the single cell. Primer concentrations in pmoles are as follows: -
AMEL 4
THO1 12
D21S11 8
D18S51 4 FGA 4
D3S1358 4
D5S818 8
D7S820 8
CSF1PO 8 D13S631 6
D13S317 6
D18S851 5
DYS14 4
D13S258 6 D21S1412 10
TPOX 2
Genetic identification of an isolated fetal cell from PAP smear using nine primer pair multiplex PCR The electropherogram in Figure 7 shows the result of amplification of nine (9) genetic markers by multiplex PCR amplification from a single fetal cell isolated from a
PAP smear.In this example 9 of 9 markers amplified successfully. PCR conditions were:- a) 94°C for 2 minutes b) 94°C for 30 seconds c) 57°C for 60 seconds d) 68°C for 30 seconds e) Go to (b), 45 times f) 72°C for 10 minutes g) Hold at 4°C
Isolated single cell samples were fixed then lysed by alkaline lysis using standard techniques before PCR processing.
PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research,
Geneworks). Master-mix for the PCR was made using AccuPrime Taq (Invitrogen); lx
PCR buffer and 1.5mM Magnesium Chloride; dNTP's were added to reach 0.2mM concentration. Primers were added as described. The master-mix was made up to 24μl using Milli-Q sterilised water.
The concentration of each set of primer was:- Marker pmol/rxn
AMEL 4
THO 12.0 D21S11 8
D18S51 4
FGA 4.0
D3S1358 4 D5S818 8 CSF 8
D7 8
Genetic identification of an isolated fetal cell from PAP smear using eleven primer pair multiplex PCR The results of multiplex amplification of eleven, respective genetic markers from a single fetal cell using eleven primer pairs are shown in Figure 8. In this example 11 of 11 markers amplified successfully. The primer concentrations were:-
Marker pmoles
AMEL 3.5
THO 6.0 D21S11 16.0
D18S51 16.0
VWA 30.0
FGA 6.0
D3S1358 10.0 D5S818 8.0
D7S820 10.0
CSF1PO 8.0
TPOX 3.0
Each single cell was treated with 5 μL of 0.624 mg/ml Proteinase K. The single cells were then subjected to the following heating program: a) 50°C for 30 minutes b) 95°C for 15 minutes
The cells were then ready for master-mix to be added and subsequent thermal cycling conditions as follows:-. a) 95°C for 1 minutes b) 94°C for 40 seconds c) 57°C for 60 seconds d) 72°C for 40 seconds e) Go to (b), 44 times f) Hold at 4°C.
Single cell PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJresearch, Geneworks). Master-mix for the PCR was made using 1.2 U of Hot Star Taq (Quiagen) per reaction in all single cell, lx PCR buffer (which contains 1.5mM Magnesium Chloride) were added (provided with the Hot star taq). dNTP's were added to reach 0.2mM concentration. Primers were added as described above. The master-mix was made up to 17.5ul using Milli-Q sterilised water. Single cells were picked using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl) Approximately 1 μL of PBS was drawn with the single cellll.
Heterozygosity of twins (DNA fingerprinting of twins in utero at 12 weeks gestation) using 9 primer multiplex PCR
The results of multiplex PCR analysis of identical (monozygotic) twins are summarized in Table 6 and Figure 9; the results of multiplex PCR analysis of non- identical (dizygotic) twins are summarized in Table 7 and Figure lO.In this example 9 of 9 markers amplified successfully. PCR cycling parameters were:- a) 95°C for 10 minutes b) 94°C for 60 seconds c) 57°C for 60 seconds d) 72°C for 60 seconds e) Go to (b), 44 times f) 72°C for 10 minutes g) Hold at 4°C
Samples from a limited amount of amniotic fluid were added to lul of lysis buffer, heated to 65°C for 10 minutes, lul of neutralising buffer was then added. PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research,
Geneworks). Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen). lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24μl using Milli-Q sterilised water. The primer concentrations were:-
Marker pmol/rxn
AMEL 3.5
THO 6.0
D21S11 16.0 D18S51 13.0
FGA 6.0 D3S1358 1.6
D5S818 1.5
CSF 12.0
TPOX 0.4 Genetic identification of single amniotic cell samples
The electropherogram in Figure 11 shows a 9 primer set multiplex from a single amniotic cell that has a 1 in 9 billion chance of two individuals having the same genetic fingerprint.
PCR cycling programme a) 95°C for 10 minutes b) 94°C for 60 seconds c) 57°C for 60 seconds d) 72°C for 60 seconds e) Go to (b), 44 times f) 72°C for 10 minutes g) Hold at4°C
Samples were added to lul of lysis buffer, heated to 65°C for 10 minutes, lul of neutralising buffer was then added.
PCR was conducted in 0.2 ml tubes on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units Hot Start taq (Qiagen) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's < were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24μl using Milli-Q sterilised water. Single cells were picked using a drawn glass pipette whilst spread in a 30mm plastic Petri dish in Phosphate buffered saline (PBS) (without Magnesium) (Gibco Brl), - lμl of PBS drawn with the single cell.
PCR primer concentrations were:- Marker pmol/rxn
AMEL 3.5 THO 6.0
D21S11 16.0 D18S51 13.0
FGA 6.0
D3S1358 1.6
D5S818 1.5 CSF 12.0
TPOX 0.4
11 primer multiplex PCR amplification from hair shaft As shown in Figure 12, DNA profiles were successfully obtained from hair roots using 11 primer sets to amplify the genetic markers listed hereinafter. The primer concentrations were:-
Marker pmol/rxn
AMEL 3.5
THO 6.0
D21S11 16.0 D18S51 16.0
VWA 30.0
FGA 6.0
D3S1358 10.0
D5S818 10.0 D7S820 10.0
CSF 13.0
TPOX 3.0
Simultaneous diagnosis and confirmation of chromosome status Figure 13 shows a amniotic low copy number sample subjected to 10 primer set multiplex. The PCR amplified genetic markers were AMEL, D13S631, D18S851, DYS14, D18S391, D13S317, D21S11, D13S258, D18S51, D21S1412 PCR cycling parameters were:- a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to b, 39 times f. 72°C for 10 minutes g. Hold at 4°C.
An amniotic cell suspension was added at 1.5μl (Stored in PBS) and run in a 96 well 200μl plate on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using 1.2 units of Amplitaq Gold (Applied Biosystems) in all single cell and amniotic samples unless otherwise stated, lx PCR buffer and 1.5mM Magnesium Chloride were added. dNTP's were added to reach 0.2mM concentration. Primers were added as described below. The master-mix was made up to 24μl using Milli-Q sterilised water.
Primer concentrations: - AMEL 1.4 pmole
D13S631 6 pmole D18S851 5 pmole D13S258 6 pmole
D18S391 11 pmole
DYS14 4 pmole
D21S11 8 pmole
D18S51 3 pmole D21S1412 12 pmole
D13S317 8 pmole
Increasing the number of simultaneous genetic diagnoses Application of multiple genetic tests according to the invention may be applied, for example, to genetic analysis such as prenatal diagnosis using nucleic acids from isolated fetal cells such as from PAP smears, amniotic fluid or other nucleic acids such as free fetal DNA in maternal blood supply.
Figure 14 shows an electropherogram of a 10 primer set multiplex PCR consisting of simultaneous detection of:
1. Single gene defect status (cystic fibrosis Delta 508 marker) 2. Sexing status (male)
3. Trisomy status (trisomy detection) 4. DNA fingerprint
The DNA was obtained using PCR on a single buccal cell This PCR contains CF1, AMEL, D13S631, D18S851, DYS14, D13S391, D13S317, D21S11, D13S258 & D18S51 Primer concentrations were (per reaction): -
AMEL 1.4 pmole
D13S631 6 pmole
D18S851 5 pmole
D13S258 6 pmole D18S51 2.6 pmole
DYS14 4 pmole
D21S11 9 pmole
D18S391 8 pmole
D13S317 5 pmole CF 5 pmol
PCR cycling parameters were:- a. 95°C for 15 minutes b. 94°C for 30 seconds c. 59°C for 45 seconds d. 72°C for 60 seconds e. Go to 2, 39 times f. 72°C for 10 minutes g. Hold at 4°C.
The single buccal cell was added at 1.5μl (Stored in PBS) and run on a PTC 200 DNA engine (MJ research, Geneworks). Master-mix for the PCR was made using
1.2 units of Amplitaq Gold (Applied Biosystems).. lx PCR buffer and 1.5mM
Magnesium Chloride were added . dNTP's were added to reach 0.2mM concentration.
Primers were added as described for each individual case. The master-mix was made up to 24μl using Milli-Q sterilised water Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scopes of the present invention. All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.
TABLE 1
STR database marker ALIAS GenBank No. Position heter PRIMER SEQUENCE
D13S241 UT556 L17673 13pter 0.83 CCA GGC ACT TTG GGA GGC TG ACC CAC TGT ATC CTG GGC A
D13S242 UT557 L18329 13q21.2 0.83 ATT GCA CCC CAT CCT GGG TCC TTT TCC TAC CAT TTG CAT
D13S243 UT558 L18330 13cen-13ql2.1 0.75 ACT GTA CTT CTG CCT GGG C TTT TGT AAT GCC TCA ACC ATG
D13S248 UT1213 L15541 13q32-13q34 0.83 ACT TAA ATG TCC ATC AAT AAA T TGA TTG GCT TTT TTT ACT TAC
D13S251 UT1329 L16338 13q31-13q32 0.75 CAC ATA GCT TAT TGT TGT TGC GTT ATC TGT GAG CAA ATA CAG
D13S253 UT1378 L16396 13q22-13q32 0.75 CTC AAG GGA TGT TAA CAC AC AGG AGG AAA AAG TGG AGA AG
D13S254 UT1585 L18690 13q31-13q32 0.86 TGA ACT CCG GCC TGG GTG A TTT TGG AGC TGG GGA TGT C
D13S256 UT2120 L17977 13ql4.1-13q22 0.91 CCT GGG CAA CAA GAG CAA A AGC AGA GAG ACA TAA TTG TG
D13S257 UT2119 L18729 13ql4.1-13q21.1 0.86 CAA CAA GAG CAA AAC TCC AT AAG CAC ATA AGT TGG TAT GAA
D13S258 UT2413 L18095 13q21.2-13q31 0.88 ACC TGC CAA ATT TTA CCA GG GAC AGA GAG AGG GAA TAA ACC
D13S303 UT936 L31309 13q22-13q31 0.75 ACA TCG CTC CTT ACC CCA TC TGT ACC CAT TAA CCA TCC CCA
D13S631 UT7403 L18392 13q31-13q32 0.94 GGC AAC AAG AGC AAA ACT CT TAG CCC TCA CCA TGA TTG G
D18S51 UT574 LI 8333 18q21.33-18q21.33 0.86 GAG CCA TGT TCA TGC CAC TG CAA ACC CGA CTA CCA GCA AC
D18S378 UT485 L16262 18P11.22-18P11.22 0.75 AGC CTG GGT GAC AGA GCA A ACA GGG AAA GCT GGG GGA T
D18S382 UT600 L16292 18pter-18pter 0.8 CAT CCA TCC ATC CTT CCA C TGT GCT GGT ATT ACA GGC G
D18S386 UT754 LI 8400 18q22.1-18q22.2 0.88 TCA GGA GAA TCA CTT GGA AC TCC ATG AAG TAG CTA AGC AG
D18S390 UT1227 L15542 18q22.3-18q23 0.75 TAA CCA AAG CAA ATC CCT GG CAC TTA CAC TGT TAT CCT GG
D18S391 UT1302 L16384 18pter-18pll.22 0.75 CTG GTT TTC GTC TTG AGA AG CAC TAT TCC CAT CTG AGT CA
D18S814 UT1248 L17776 18pter-18pter 0.73 CTT CCC TGG GTA TCA AGA CT TCC CAC TAT ATG TAT GTT CAC C
D18S815 UT1438 L17819 18pter-18qter 0.75 GGC TGA GAC AGG AGA ATC AC CTC ACC AGG ATT TCC TTG C
D18S819 UT7251 L30411 18pter-18qter 0.75 ACC ACA GTT ACT AAG ATG TAA GCC TCC AGA AAA AAT TTC CA
SHGC
D18S851 4561 G08002 18pter-18qter CTG TCC TCT AGG CTC ATT TAG C TTA TGA AGC AGT GAT GCC AA
D21S11 VS17T3 M84567 21q21-21q21 0.9 GTG AGT CAA TTC CCC AAG GTT GTA TTA GTC AAT GTT CTC C
D21S1240 UT656 LI 8360 21pter-21qter 0.5 GAG ACG GTA GGA AAA GGA G AGC CAA GTT CGA GCC ACT G
D21S1244 UT761 L16331 21q21-21q22.1 0.8 GTC CCC ATA TTG ATA AAC TAT T ATG AAT AGG GGA TAT GCT GG
D21S1413 UT7582 L30513 21pter-21pter 0.875 TTG CAG GGA AAC CAC AGT T TCC TTG GAA TAA ATT CCC GG
D21S1412 UT6930 L29680 21pter-21pter 0.8 CGG AGG TTG CAG TGA GTT G GGG AAG GCT ATG GAG GAG A
D21S1411 UT1355 L17803 21pter-21pter 0.933 ATG ATG AAT GCA TAG ATG GAT G AAT GTG TGT CCT TCC AGG C
PENTA E PAUL1 AC027004 21q 0.88 TCC AGC CTA GGT GAC AGA GC TGC CTA AAC CTA TGG TCA TAA CG
AMEL M55418 Xp22.31-p22.1 n/a CCC TGG GCT CTG TAA AGA ATA GTG ATC AGA GCT TAA ACT GGG AAG CTG
HUMTHO D00269 llpl5-15.5 0.76 GCT TCC GAG TGC AGG TCA CA CAG CTG CCC TAG TCA GCA C
TPOX M68651 2p23-2pter 0.65 CAC TAG CAC CCA GAA CCG TC CCT TGT CAG CGT TTA TTT GCC CCC TAG TGG ATG ATA AGA ATA ATC
VWA M25858 12pl2-pter 0.83 AGT ATG
GGA CAG ATG ATA AAT ACA TAG GAT GGA TGG
D3S1358 11449919 3p 0.78 ACT GCA GTC CAA TCT GGG T ATG AAA TCA ACA GAG GCT TG
D5S818 G08446 5q21-q31 0.71 GGG TGA TTT TCC TCT TTG GT TGA TTC CAA TCA TAG CCA CA
D7S820 G08616 7q 0.79 TGT CAT AGT TTA GAA CGA ACT AAC G CTG AGG TAT CAA AAA CTC AGA GG
CSF1PO U63963 X14720 5q33.3-34 0.78 AAC CTG AGT CTG CCA AGG ACT AGC TTC CAC ACA CCA CTG GCC ATC TTC
FGA M64982 4q28 0.86 GCC CCA TAG GTT TTG AAC TCA TGA TTT GTC TGT AAT TGC CAG C
D13S317 G09017 13q22-q31 0.71 ACA GAA GTC TGG GAT GTG GA GCC CAA AAA GAC AGA CAG AA
DYS14 n/a CTT TCC ACA GCC ACA TTT GTC CAT CCA GAG CGT CCC TGG CTT
CF GTT TTC CTG GAT TAT GCC TGG GCA GTT GGC ATG CTT TGA TGA CGC TTC
D16S539 GAT CCC AAG CTC TTC CTC TT ACG TTT GTG TGT GCA TCT GT
D16S690 GCA CAG CTT CCT GAT CTG A TCA CAC AAC CCA CAG AGA A D22S417 CCT GGG AAG TTA AGA CTG C TCT ACC GCT TAT TTC TTC CCT D22S526 AGA GCA AGA CTC TGT CTC AAC A TTC TCC TTC ACT TTC TGC CAT G
TABLE 2
Figure imgf000042_0001
TABLE 3
Figure imgf000043_0001
TABLE 4
Figure imgf000043_0002
TABLE 5
Figure imgf000044_0001
TABLE 6
Figure imgf000045_0002
Table 6b
Figure imgf000045_0001
TABLE 7
Figure imgf000046_0001

Claims

1. A method of selecting a plurality of genetic markers as targets for nucleic acid sequence amplification, said method including the step of selecting each of said plurality of genetic markers according to a heterozygosity index, wherein said heterozygosity index is 0.5 or greater.
2. The method of Claim 1, wherein said heterozygosity index is 0.7 or greater.
3. The method of Claim 1, wherein said heterozygosity index is 0.9 or greater.
4. The method of Claim 1 further including the step of selecting one or more primers for each respective said genetic marker so that upon amplification of said genetic marker using said one or more primers, a resultant amplification product has a molecular size in the range 50-3000 bp.
5. The method of Claim 4, the molecular size is in the range 50-1000 bp.
6. The method of Claim 4, wherein the molecular size is in the range 80-500 bp.
7. The method of Claim 4, wherein the molecular size is in the range 100-400 bp.
8. The method of Claim 1, wherein each of said genetic markers is a short tandem repeat (STR).
9. A method of nucleic acid sequence amplification, said method including the step of using a nucleic acid sequence amplification technique and at least nine primer pairs in combination to amplify a plurality of respective genetic markers from a limiting amount of nucleic acid sample.
10. The method of Claim 9, wherein at least ten primer pairs are used to amplify said respective genetic markers.
11. The method of Claim 9, wherein at least eleven primer pairs to amplify said respective genetic markers.
12. The method of Claim 9, wherein at least twelve primer pairs are used to amplify said respective genetic markers.
13. The method of Claim 9, wherein at least thirteen primer pairs are used to amplify said respective genetic markers.
14. The method of Claim 9, wherein at least fourteen primer pairs are used to amplify said respective genetic markers.
15. The method of Claim 9, wherein at least fifteen primer pairs are used to amplify said respective genetic markers.
16. The method of Claim 9, wherein sixteen primer pairs are used in combination to amplify said respective genetic markers.
17. The method of Claim 9 wherein the nucleic acid sample has less than 1 ng of DNA.
18. The method of Claim 9 wherein the nucleic acid sample has less than 500 pg of DNA.
19. The method of Claim 9, wherein the nucleic acid sample has less than 200 pg of DNA.
20. The method of Claim 9, wherein the nucleic acid sample has 3-6 pg of target DNA.
21. The method of Claim 9, wherein said nucleic acid sample comprises DNA isolated from less than 200 cells, or an equivalent amount of DNA.
22. The method of Claim 9, wherein said nucleic acid sample comprises DNA isolated from less than 100 cells or an equivalent amount of DNA
23. The method of Claim 9, wherein said nucleic acid sample comprises DNA isolated from less than 50 cells or an equivalent amount of DNA
24. The method of Claim 9, wherein said nucleic acid sample comprises DNA isolated from less than 10 cells or an equivalent amount of DNA
25. The method of Claim 9 wherein the nucleic acid sample comprises DNA isolated from a single cell, or an equivalent amount of DNA.
26. The method of Claim 25 wherein the single cell is a fetal cell.
27. The method of Claim 26, wherein the fetal cell is isolated from a PAP smear.
28. The method of Claim 26, wherein the fetal cell is obtained by CVS, amniocentesis or is obtained from fetal blood.
29. The method of Claim 25 wherein the single cell is a human embryonic cell.
30. The method of Claim 25 wherein the single cell is a haploid cell.
31. The method of Claim 30, wherein the haploid cell is a spermatozoa.
32. The method of Claim 25 wherein the single cell is a human epithelial cell.
33. The method of Claim 25, wherein the single cell is obtained from a non-human mammal.
34. The method of Claim 9 wherein nucleic acid sequence amplification is PCR.
35. The method of Claim 34 wherein PCR is fluorescent multiplex PCR.
36. The method of Claim 9, wherein the primers are selected from the group consisting of SEQ ID NOS: 1-92.
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