Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING 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
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6858—Allele-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.
• 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

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