US20040137504A1 - Multiplex amplification of short tandem repeat loci - Google Patents

Multiplex amplification of short tandem repeat loci Download PDF

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US20040137504A1
US20040137504A1 US10/769,579 US76957904A US2004137504A1 US 20040137504 A1 US20040137504 A1 US 20040137504A1 US 76957904 A US76957904 A US 76957904A US 2004137504 A1 US2004137504 A1 US 2004137504A1
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loci
genomic dna
humth01
humhprtb
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James Schumm
Cynthia Sprecher
Ann Lins
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Promega Corp
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    • 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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention is generally directed to the detection of genetic markers in a genomic system.
  • the present invention is more specifically directed to the simultaneous amplification of multiple distinct polymorphic genetic loci using the polymerase chain reaction or other amplification systems to determine in one reaction the alleles of each locus contained within the multiplex system.
  • STRs polymorphic short tandem repeats
  • STR loci consist of short, repetitive sequence elements of 3 to 7 base pairs in length. It is estimated that there are 2,000,000 expected trimeric and tetrameric STRs present as frequently as once every 15 kilobases (kb) in the human genome (Edwards et al. 1991; Beckmann and Weber 1992). Nearly half of the STR loci studied by Edwards et al. (1991) are polymorphic, which provides a rich source of genetic markers. Variation in the number of repeat units at a particular locus is responsible for the observed polymorphism reminiscent of VNTR loci (Nakamura et al.
  • STR loci are extremely useful markers for human identification, paternity testing and genetic mapping.
  • STR loci may be amplified via the polymerase chain reaction (PCR) by employing specific primer sequences identified in the regions flanking the tandem repeat.
  • PCR polymerase chain reaction
  • Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another following electrophoretic separation by any suitable detection method including radioactivity, fluorescence, silver stain, and color.
  • Cystic Fibrosis Estivill et al. 1991, Fortina et al. 1992, Ferrie et al. 1992, Morral and Estivill, 1992
  • Retinoblasma Retinoblasma
  • Multiplex amplification of polymorphic microsatellite markers (Clemens et al. 1991, Schwartz et al. 1992, Huang et al. 1992) and even STR markers (Edwards et al. 1992, Kimpton et al. 1993, Hammond et al. 1994) have been described.
  • amplified products are generally separated by one of several methods of electrophoresis known to those skilled in the art.
  • Several well-known methods of detection of the amplified products have also been described. While ethidium bromide staining of amplified fragments is employed in some cases, in others it is preferred to use methods which label only one of the two strands of the amplified material. Examples of this include radioactive or fluorescent labeling of one of the two primers prior to the amplification of a locus.
  • One of the more sophisticated approaches to detection is the use of different fluorescent labels to allow detection of amplified materials representing different loci, but existing in the same space following electrophoresis.
  • the products of the different loci are differentiated with the use of filters, which allow visualization of one fluorescent label at a time.
  • Ballabio et al. (1991) disclose a single tube multiplex allele specific PCR test using two different dye-tagged fluorescent primers for detection of the AF508 cystic fibrosis mutation.
  • the present invention is directed to a method of simultaneously analyzing or determining the alleles present at each individual locus of each multiplex.
  • This method comprises the steps of (1) obtaining at least one Genomic DNA sample to be analyzed, wherein the Genomic DNA sample has at least two loci which can be amplified together; (2) amplifying the STR sequences in the DNA sample; and (3) detecting the amplified materials in a fashion which reveals the polymorphic nature of the systems employed.
  • the present invention is also directed to a method of simultaneously analyzing multiple STR sequences wherein at least one of the loci is selected from the group consisting of: HUMCSFLPO, HUMTPOX, HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B), HUMLIPOL, HSAC04 (ACTBP2), HUMCYP19, HUMPLA2A1, HUMAPOA2, HUMCD4, HUMF13A01 and HUMMYOPK (Myotonic).
  • the present invention is directed to a method of simultaneously analyzing multiple STR sequences in the following groups of loci: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX and HUMTH01; HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUM
  • the present invention provides a high throughput method for the detection and analysis of polymorphic genetic markers using specific combinations of loci and specified conditions.
  • the process can be used in laboratories which have only a power supply and a standard apparatus for/polyacrylamide gel electrophoresis or those which have the latest in equipment for fluorescent gel scanning, e.g., FluorImagerTM-575 (Molecular Dynamics, Sunnyvale, Calif.).
  • FluorImagerTM-575 Molecular Dynamics, Sunnyvale, Calif.
  • the approach as specified in the present invention produces a savings in time, labor and materials in the analysis of loci contained within the multiplexes.
  • the process of the present invention includes all the requisite primers, allowing between two and four or more loci to be amplified together in one amplification tube instead of amplifying each locus independently.
  • the present invention has specific use in the field of forensic analysis, paternity determination, monitoring of bone marrow transplantation, linkage mapping, and detection of genetic diseases and cancers.
  • FIG. 1 is a photograph illustrating the simultaneous amplification of three loci: HUMCSF1PO, HUMTPOX and HUMTH01, with the amplified products of each locus shown migrating next to the corresponding allelic ladder for ease of interpretation in Example 1.
  • FIG. 2 is a computer image showing the fluorescent detection of multiplex amplification of the loci HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31 as detected with a FluorImagerTM-575 (Molecular Dynamics, Sunnyvale, Calif.) in Example 2.
  • FIG. 3 is a photograph showing the silver stain detection of the multiplex amplification in Example 3.
  • FIG. 4 is a computer image showing the fluorescent detection of multiplex amplification in Example 4.
  • FIG. 5 is a photograph showing the silver stain detection of the multiplex amplification in Example 5.
  • FIG. 6 is a photograph showing the silver stain detection of the multiplex amplification in Example 6.
  • FIG. 7 is a photograph showing the silver stain detection of the multiplex amplification in Example 7.
  • FIG. 8 is a photograph showing the silver stain detection of the multiplex amplification in Example 8.
  • FIG. 9 is a photograph showing the silver stain detection of the multiplex amplification in Example 9.
  • FIG. 10 is a photograph showing the silver stain detection of the multiplex amplification in Example 10.
  • FIG. 11 is a photograph showing the silver stain detection of the multiplex amplification in Example 11.
  • FIG. 12 is a photograph showing the silver stain detection of the multiplex amplification in Example 12.
  • FIG. 13 is a photograph showing the silver stain detection of the multiplex amplification in Example 13.
  • FIG. 14 is a photograph showing the silver stain detection of the multiplex amplification in Example 14.
  • FIG. 1S is a photograph showing the silver stain detection of the multiplex amplification in Example 15.
  • FIG. 16 is a photograph showing the silver stain detection of the multiplex amplification in Example 16.
  • FIG. 17 is a photograph showing the silver stain detection of the multiplex amplification in Example 17.
  • FIG. 18 is a photograph showing the silver stain detection of the multiplex amplification in Example 18.
  • FIG. 19 is a photograph showing the silver stain detection of the multiplex amplification in Example 19.
  • FIG. 20 is a photograph showing the silver stain detection of the multiplex amplification in Example 20.
  • FIG. 21 is a photograph showing the silver stain detection of the multiplex amplification in Example 21.
  • FIG. 22 is a photograph showing the silver stain detection of the multiplex amplification in Example 22.
  • FIG. 23 is a photograph showing the silver stain detection of the multiplex amplification in Example 23.
  • FIG. 25 is a photograph showing the silver stain detection of the multiplex amplification in Example 25.
  • FIG. 26 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 26.
  • FIG. 27 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 27.
  • FIG. 28 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 28.
  • FIG. 29 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 29.
  • FIG. 30 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 30.
  • FIG. 31 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 31.
  • FIG. 32 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 32.
  • Allelic ladder a standard size marker consisting of amplified alleles from the locus.
  • Allele a genetic variation/associated with a segment of Genomic DNA, i.e., one of two or more alternate forms of a DNA sequence occupying the same locus.
  • Biochemical nomenclature standard biochemical nomenclature is used herein in which the nucleotide bases are designated as adenine (A); thymine (T); guanine (G); and cytosine (C).
  • Corresponding nucleotides are, for example, deoxyguanosine-5′-triphosphate (dGTP).
  • Genomic DNA polymorphism the condition in which two or more different nucleotide sequences coexist in the same interbreeding population in a Genomic DNA sequence.
  • Locus (or genetic locus): a specific position on a chromosome. Alleles of a locus are located at identical sites on homologous chromosomes.
  • Locus-specific primer a primer that specifically hybridizes with a portion of the stated locus or its complementary strand, at least for one allele of the locus, and does not hybridize efficiently with other Genomic DNA sequences under the conditions used in the amplification method.
  • PCR Polymerase chain reaction
  • PIC Polymorphism information content
  • Primary reaction initial reaction using the purified human genomic DNA as template for the PCR.
  • Primers two single-stranded oligonucleotides or DNA fragments which hybridize with opposing strands of a locus such that the 3′ termini of the primers are in closest proximity.
  • Primer pair two primers including primer 1 that hybridizes to a single strand at one end of the Genomic DNA sequence to be amplified and primer 2 that hybridizes with the other end on the complementary strand of the Genomic DNA sequence to be amplified.
  • Primer site the area of the target Genomic DNA to which a primer hybridizes.
  • Secondary reaction reamplification with the same or different primer pair using a dilution of the primary reaction as template for the PCR.
  • an appropriate set of loci, primers, and amplification protocols Prior to constructing the multiplex system, an appropriate set of loci, primers, and amplification protocols must be selected such that amplification generates fragments such that alleles of the various loci do not overlap in size or, when such overlap occurs, fragments representing different loci are detectable by separate means.
  • the selected loci must be compatible for use with a single amplification-protocol.
  • the specific combinations of loci described herein are unique in this application. Combinations of loci may be rejected for either of these reasons, or because, in combination, one or more of the loci do not produce adequate product yield, or fragments which do not represent authentic alleles are produced in this reaction.
  • the primers must also be designed so that the size of the resulting amplification products differ in length, thereby facilitating assignment of alleles to individual loci during detection. Inappropriate selection of primers can produce several undesirable effects such as lack of amplification, amplification at multiple sites, primer dimer formation, undesirable interaction of primer sequences from different loci, production of alleles from one locus which overlap with alleles from another, or requirement for amplification conditions or protocols for the different loci which are incompatible in a multiplex.
  • the synthesis of the primers is conducted by procedures known to those skilled in the art.
  • a multiplex containing two loci may be used as a core to create multiplexes containing more than two loci. New combinations are created including the first two loci.
  • the core multiplex containing loci HUMTH01 and HUMCSF1PO was used to generate derivative multiplexes of HUMTH01, HUMCSFLPO, and HUMTPOX; HUMTH01, HUMCSF1PO, and HUMCD4; HUMTH01, HUMCSF1PO, and HUMVWFA31; HUMTH01, HUMCSF1PO, HUMVWFA31, and HUMTPOX; and HUMTH01, HUMCSFLPO, HUMCD4, and HUMTPOX.
  • Many other derivative multiplexes can be generated based upon a working multiplex.
  • the derivative multiplexes are, in some sense, routine extensions of the core multiplex.
  • Genomic DNA preparation which are compatible with the amplification process for a single locus should be appropriate for multiplex amplification. Many examples of preparation methods have been described in the literature (Patel et al. 1984, Gill et al. 1985). Genomic DNA concentrations are measured fluorometrically (Brunk et al. 1979).
  • locus-specific primers include a number of nucleotides which, under the conditions used in the hybridization, are sufficient to hybridize with an allele of the locus to be amplified and to be essentially free from amplification of alleles of other loci.
  • U.S. Pat. No. 5,192,659 to Simons which is incorporated herein by reference for a more detailed description of locus-specific primers.
  • products are then separated by electrophoresis, e.g., denaturing polyacrylamide gel electrophoresis (Sambrook et al., 1989).
  • electrophoresis e.g., denaturing polyacrylamide gel electrophoresis (Sambrook et al., 1989).
  • Preferred gel preparation and electrophoresis procedures are conducted as described in Example 1. Fragment separation occurs based on size and charge of the sample.
  • Genomic DNA is then detected by, e.g., silver staining (Bassam et al. 1991).
  • silver staining Bassam et al. 1991
  • the products are detected by means available to detect these reporters as known to those skilled in the art.
  • Amplified materials may be detected using any of a number of reporters including, e.g., silver staining, radioisotopes, fluorescers, chemiluminescers and enzymes in combination with detectable substrates.
  • Genomic DNA samples containing amplified alleles are preferably compared with a size standard such as a Genomic DNA marker or locus-specific allelic ladder to determine the alleles present at each locus within the sample.
  • a size standard such as a Genomic DNA marker or locus-specific allelic ladder to determine the alleles present at each locus within the sample.
  • the preferred size marker for evaluation of a multiplex amplification containing two or more polymorphic STR loci consists of a combination of allelic ladders for the loci being evaluated.
  • the preferred size marker for evaluation of a multiplex amplification containing two or more polymorphic STR loci which are generated using fluorescently-labeled primers for each locus consists of a combination of fluorescently-labeled allelic ladders for the loci being evaluated.
  • allelic ladders for individual loci, they may be mixed and loaded for gel electrophoresis at the same time as the loading of amplified samples occurs. Each allelic ladder co-migrates with alleles in the sample from the corresponding locus.
  • a permanent record of the data can be generated with the use of electrophoresis duplicating film (STR systems manual #TMD004, Promega Corporation, Madison, Wis.).
  • a filter prohibits passage of the original light, but allows collection of the emitted light by a fiber optic collector.
  • a second filter selected by the user may be inserted between the fiber optic collector and the photomultiplier, allowing detection of specific wavelength bands (or colors) with each scan.
  • the image has an overall cleaner appearance than that obtained with the silver stain for three reasons.
  • Third, some artifact bands of the PCR reaction are plentiful, but contain very little primer.
  • kits that utilize the process described.
  • a basic kit includes a container having a locus-specific primer pair (or alternately separate containers containing each primer of a primer pair) for each locus.
  • the kit also includes instructions for use.
  • Other ingredients may include an allelic ladder directed to each of the specified loci, a sufficient quantity of enzyme for amplification, amplification buffer to facilitate the amplification, loading solution for preparation of the amplified material for gel electrophoresis, human genomic DNA as a control to test that the system is working well, a size marker to insure that materials migrate as anticipated in the gel, and a protocol and manual to educate the user and to limit error in use.
  • the amounts of the various reagents in the kits can be varied depending upon a number of factors, such as the optimum sensitivity of the process.
  • the instructions for use are suitable to enable any analyst to carry out the desired test. It is within the scope of this invention to provide test kits for use in manual applications or test kits for use with automated detectors or analyzers.
  • Genomic DNA isolation and quantitation were performed essentially as described by Puers et al., 1993. These methods are generally known to those skilled in the art and are preferred, but not required, for application of the invention.
  • Amplification products were separated by electrophoresis through a 0.4 mm thick 4% denaturing polyacrylamide gel (19:1 ratio of acrylamide to bis-acrylamide) which contained 7 M urea (Sambrook et al., 1989) and was chemically cross-linked to one glass plate (Kobayashi, 1988). Genomic DNA samples were mixed with 3 ⁇ l loading solution (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol), denatured at 95° C. for 2 min., and chilled on ice prior to loading.
  • Electrophoresis was performed at 60 W in 0.5 ⁇ TBE for 1-2 hrs.
  • the Genomic DNA was detected by silver staining (Bassam et al., 1991). Permanent images were obtained by exposure to Electrophoresis Duplicating Films (EDF, Kodak, Cat. No. 809 6232).
  • EDF Electrophoresis Duplicating Films
  • detection can be performed by fluorescent scanning (Schumm et al., 1994) or radioactive detection (Hammond et al., 1994).
  • a Genomic DNA template (three Genomic DNA samples) was amplified at the individual loci HUMCSFLPO, HUMTPOX, and HUMTH01 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 50 ⁇ l volumes using 25 ng template, 0.03 U Taq Genomic DNA Polymerase/ ⁇ l 1 ⁇ STR Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100, 1.5 mM MgCl2 and 200 ⁇ M each of dATP, dCTP, dGTP and dTTP), and using a Thermal Cycler 480 (Perkin Elmer Cetus).
  • Amplification protocol 1 (96° C. for 2 min., then 10 cycles of 94° C. for 1 min., 64° C. for 1 min., and 70° C. for 1.5 min., followed by 20 cycles of 90° C. for 1 min., 64° C. for 1 min., 70° C. for 1.5 min.) was employed.
  • each amplification primers were used in combination, including 0.2 ⁇ M each HUMCSF1PO primers 1 [SEQ. ID. 5] and 2 [SEQ. ID. 6], 0.2 ⁇ M each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 0.6 ⁇ M each HUMTH01 primers 1 [SEQ. ID. 27] and 2 [SEQ. ID. 28].
  • Amplified products were separated by denaturing acrylamide gel electrophoresis on a 40 cm gel for 60-90 min. at 60 W and products were visualized by silver stain analysis according the protocol of Bassam et al. (1991).
  • FIG. 1 reveals the silver stain detection of the multiplex amplification.
  • Lanes 2, 3, and 5 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSFLPO, HUMTPOX, and HUMTH01.
  • Lanes 1, 4, and 7 contain allelic ladders for the three loci and lane 6 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the individual loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMVWFA31 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 25 ⁇ l volumes using 25 ng template, 0.04 U Taq Genomic DNA Polymerase/ ⁇ l, 1 ⁇ STR Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100, 1.5 mM MgCl2 and 200 ⁇ M each of DATP, dCTP, dGTP and dTTP), and using a Thermal Cycler 480 (Perkin Elmer Cetus).
  • Amplification protocol 1 as described in Example 1, was employed. Eight amplification primers were used in combination, including 1 ⁇ M each HUMCSF1PO primer 2 [SEQ. ID. 5] and fluorescein-labeled primer 1 [SEQ. ID. 5], 0.15 ⁇ M each HUMTPOX primer 1 [SEQ. ID. 29] and fluorescein-labeled primer 2 [SEQ. ID. 30], 0.2 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 271, and 1M each HUMVWFA31 primer 1 [SEQ. ID. 31] and fluorescein-labeled primer 2 [SEQ. ID. 32].
  • FIG. 2 is a computer image of a FluorImager scan. Lanes 2-7 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMVWFA31. Lane 1 contains allelic ladders for the 4 loci.
  • a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMVWFA31 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 25 ⁇ l volumes using 25 ng template, 0.03 U Taq Genomic DNA Polymerase/ ⁇ l, 1 ⁇ STR Buffer (described in example 1), and a Thermal Cycler 480 (Perkin Elmer Cetus).
  • Amplification protocol 2 (96° C. for 2 min., then 10 cycles of 94° C. for 1 min., 60° C. for 1 min., and 70° C. for 1.5 min., followed by 20 cycles of 90° C. for 1 min., 64° C. for 1 min., 70° C.
  • Lanes 2-6 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMVWFA31. Lanes 1 and 7 contain allelic ladders for the 3 loci.
  • Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B), and HUMLIPOL simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 2 using amplification protocol 2, as described in Example 3.
  • primers were used in combination, including 1M each HUMHPRTB primer 2 [SEQ. ID. 20] and fluorescein-labeled primer 1 [SEQ. ID. 19], 2.5 ⁇ M each HUMFESFPS primer 2 [SEQ. ID. 18] and fluorescein-labeled primer 1 [SEQ. ID. 17], 1 ⁇ M each HUMBFXIII (F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1 [SEQ. ID. 13], and 0.5 ⁇ M each HUMLIPOL primer 2 [SEQ. ID. 24] and fluorescein-labeled primer 1 [SEQ. ID. 23].
  • FIG. 4 is a computer image of a FluorImager scan.
  • Lanes 2-7 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B), and HUMLIPOL.
  • Lane 1 contains allelic ladders for the 4 loci.
  • a Genomic DNA template was amplified at the individual loci HSAC04 and HUMCYP19 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 15 ⁇ l volumes with 25 ng template, 0.01 U Taq Genomic DNA Polymerase/ ⁇ l, 1 ⁇ Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 ⁇ M each of dATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus).
  • Amplification protocol 2 as described in Example 3, was employed.
  • Amplified products were separated and detected per example 1.
  • Four primers were used in combination, including 1 ⁇ M each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], and 1 ⁇ M each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2) and HUMCYP19. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 15 ⁇ l volumes with 25 ng template, 0.02 U Taq DNA Polymerase/ ⁇ l, 1 ⁇ Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 ⁇ M each of DATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus).
  • Amplification protocol 2 as described in Example 3, was employed. Amplified products were separated and detected per example 1.
  • Six primers were used in combination, including 1 ⁇ M each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], 1 ⁇ M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26], and 1 ⁇ M each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HSAC04 (ACTBP2) and HUMFABP simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 5 using amplification protocol 2, as described in Example 3.
  • Four primers were used in combination, 1 ⁇ M each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], and 1 ⁇ M each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2) and HUMFABP. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMAPOA2, HUMCYP19, and HUMPLA2A simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 6 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMAPOA2 primers 1 [SEQ. ID. 3] and 2 [SEQ. ID. 4], 1 ⁇ M each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], and 1M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Lanes 1 and 3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMAPOA2, HUMCYP19, and HUMPLA2A1.
  • Lane 2 contains a Genomic DNA sample which failed to amplify and lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMCD4, HUMCSF1PO, and HUMTH01 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed in 50 ⁇ l volumes with 25 ng template, 0.02 U Taq Genomic DNA Polymerase/ ⁇ l, 1 ⁇ Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 ⁇ M each of DATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus).
  • a Thermal Cycler 480 Perkin Elmer Cetus
  • Amplification protocol 1 as described in Example 1, was employed. Amplified products were separated and detected as described in Example 1.
  • Six primers were used in combination, including 1 ⁇ M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10], 1 ⁇ M each HUMCSF1PO primers 1 [SEQ. ID. 5] and 2 [SEQ. ID. 6], and 1 ⁇ M each HUMTH01 primers 1 [SEQ. ID. 27] and 2 [SEQ. ID. 28].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCD4, HUMCSF1PO, and HUMTH01. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMCYP19, HUMFABP, and HUMPLA2A1 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 6 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], 1 ⁇ M each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16] and 1 ⁇ M each HUMPLA1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCYP19, HUMFABP, and HUMPLA2A1.
  • Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMCYP19, HUMHPRTB, and HUMPLA2A1 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 9 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], 1 ⁇ M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], and 1 ⁇ M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCYP19, HUMHPRTB, and HUMPLA2A1.
  • Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMF13A01 and HUMFABP simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 5 using amplification protocol 1, as described in Example 1.
  • Four primers were used in combination, including 1 ⁇ M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12], and 1 ⁇ M each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMFABP. Lane 4 displays a sample without DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMBFXIII (F13B) and HUMFESFPS simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 6 using amplification protocol 1, as described in Example 1.
  • Four primers were used in combination, including 1 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14], and 1 ⁇ M each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B) and HUMFESFPS. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMBFXIII (F13B), HUMHPRTB, and HUMPLA2A1 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 6 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14], 1M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], and 1M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B), HUMHPRTB, and HUMPLA2A1. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMF13A01, HUMFABP, and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 5 using amplification protocol 1, as described in Example 1.
  • Six primers were used in combination, including 1 ⁇ M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12], 1M each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16], and 1M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 101.
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01, HUMFABP, and HUMCD4. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMHPRTB and HUMFESFPS simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using 500-0.5 ng template, 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 2, as described in Example 3.
  • Four primers were used in combination, including 0.2 ⁇ M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20] and 1.5 ⁇ M each HUMFESFPS primers 1 [SEQ. ID. 171 and 2 [SEQ. ID.
  • Lanes 1-6 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB and HUMFESFPS-using 500, 50, 25, 5, 1 and 0.5 ng DNA template. Lane 7 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMLIPOL simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 0.4 ⁇ M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], 3 ⁇ M each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 2 ⁇ M each HUMLIPOL primers 1 [SEQ. ID. 23] and 2 [SEQ. ID. 24].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS and HUMLIPOL. Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
  • a Genomic DNA template was amplified at the loci HUMBFXIII (F13B) and HUMLIPOL Simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/pl and amplification protocol 2, as described in Example 3.
  • Four primers were used in combination, including 1 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14] and 1 ⁇ M each HUMLIPOL primers 1 [SEQ. ID. 23] and 2 [SEQ. ID. 24].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B) and HUMLIPOL.
  • a Genomic DNA template was amplified at the loci HUMHPRTB, HUMTPOX, and HUMBFXIII (F13B) simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1M each HUMHPRTB primers 1. [SEQ. ID. 19] and 2 [SEQ. ID. 20], 0.2 ⁇ M each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 2 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMTPOX, and HUMBFXIII (F13B).
  • a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMBFXIII (F13B) simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], 2 ⁇ M each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 2 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMBFXIII (F13B).
  • a Genomic DNA template was amplified at the loci HUMCSF1PO, HUMTPOX, and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using amplification protocol 1, as described in Example 1.
  • Six primers were used in combination, including 1 ⁇ M each HUMCSFLPO primers 1 [SEQ. ID. 5] and 2 (SEQ. ID. 61], 1 ⁇ M each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 1 ⁇ M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, and HUMCD4.
  • a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMMYOPK simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using amplification protocol 2, as described in Example 3.
  • Six primers were used in combination, including 1 ⁇ M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 (SEQ. ID. 20], 1M each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 1 ⁇ M each HUMMYOPK (Myotonic) primers 1 [SEQ. ID. 21] and 2 [SEQ. ID. 22].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMMYOPK (Myotonic).
  • a Genomic DNA template was amplified at the loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Eight primers were, used in combination, including 1 ⁇ M each HUMCSF1PO primers 1 (SEQ. ID. 5] and 2 (SEQ. ID. 6], 1 ⁇ M each HUMTPOX primers 1 [SEQ. ID. 291 and 2 [SEQ. ID. 30], 1 ⁇ M each HUMTH01 primers 1 (SEQ. ID. 27] and 2 [SEQ. ID. 28], and 1 ⁇ M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10].
  • FIG. 23 reveals the silver stain detection of the multiplex amplification.
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4.
  • a Genomic DNA template was amplified at the loci HUMF13A01 and HUMMYOPK (Myotonic) simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Four primers were used in combination, including 0.1 ⁇ M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12] and 1 ⁇ M each HUMMYOPK (Myotonic) primers 1 [SEQ. ID. 21] and 2 [SEQ. ID. 22].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMMYOPK (Myotonic).
  • a Genomic DNA template was amplified at the loci HUMF13A01 and HUMBFXIII (F13B) simultaneously in a single reaction vessel.
  • the PCR amplifications and other manipulations were performed as described in Example 1 using 0.03 U Taq Genomic DNA Polymerase/A1 and amplification protocol 2, as described in Example 3.
  • Four primers were used in combination, including 0.1 ⁇ M each HUMF13A01 primers 1 [SEQ. ID. 11] and 2-[SEQ. ID. 12] and 0.5 ⁇ M each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMBFXIII (F13B).
  • a Genomic DNA template was amplified at the individual loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Eight amplification primers were used in combination, including 2 ⁇ M each HUMCSFLPO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 0.5 ⁇ M each HUMTPOX primer 1 [SEQ. ID. 29] and fluorescein-labeled primer 2 [SEQ. ID.
  • FIG. 26 is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4.
  • a Genomic DNA template was amplified at the individual loci HUMCSF1PO, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Six amplification primers were used in combination, including 1 ⁇ M each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 1 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 281 and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 ⁇ M each HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID. 10].
  • FIG. 27 is a photograph of a computer image of a FluorImager scan. Lanes 1 and 2 contain DNA samples simultaneously co-amplified for the loci HUMCSFLPO, HUMTH01, and HUMCD4.
  • a Genomic DNA template was amplified at the individual loci HUMCSFLPO, HUMTH01, and HUMVWFA31 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Six amplification primers were used in combination, including 1M each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 1 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27], and 1 ⁇ M each HUMVWFA31 primer 1 [SEQ. ID. 31] and fluorescein-labeled primer 2 [SEQ. ID. 32].
  • FIG. 28 is a photograph of a computer image of a FluorImager scan. Lanes 1 and 2 contain DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTH01, and HUMVWFA31.
  • a Genomic DNA template was amplified at the individual loci HUMHPRTB, HUMBFXIII (F13B), and HUMLIPOL simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.03 U Taq Genomic DNA Polymerase/A1 and amplification protocol 2, as described in Example 3.
  • Six amplification primers were used in combination, including 1 ⁇ M each HUMHPRTB primer 2 (SEQ. ID. 20] and fluorescein-labeled primer 1 [SEQ. ID. 19], 1M each HUMBFXIII (F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1 [SEQ. ID. 13], and 1 ⁇ M each HUMLIPOL primer 2 [SEQ. ID. 24] and fluorescein-labeled primer 1 [SEQ. ID. 23].
  • FIG. 29 is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMBFXIII (F13B), and HUMLIPOL.
  • a Genomic DNA template was amplified at the individual loci HUMCSF1PO and HUMTH01 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Four amplification primers were used in combination, including 2 ⁇ M each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5] and 1 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27].
  • FIG. 30 is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSFLPO and HUMTH01.
  • a Genomic DNA template was amplified at the individual loci HUMTH01 and HUMCD4 simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Four amplification primers were used in combination, including 1 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 ⁇ M each HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID. 10].
  • FIG. 31 is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMTH01 and HUMCD4.
  • a Genomic DNA template was amplified at the individual loci HUMTH01 and HUMTPOX simultaneously in a single reaction vessel.
  • the PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and amplification protocol 1, as described in Example 1.
  • Four amplification primers were used in combination, including 1 ⁇ M each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 ⁇ M each HUMTPOX primer 2 [SEQ. ID. 30] and fluorescein-labeled primer 1 [SEQ. ID. 29].
  • FIG. 32 is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMTH01 and HUMTPOX.
  • VNTR Variariable number of tandem repeat

Abstract

The present invention is directed to the simultaneous amplification of multiple distinct genetic loci using PCR or other amplification systems to determine in one reaction the alleles of each locus contained within the multiplex.

Description

    FIELD OF THE INVENTION
  • The present invention is generally directed to the detection of genetic markers in a genomic system. The present invention is more specifically directed to the simultaneous amplification of multiple distinct polymorphic genetic loci using the polymerase chain reaction or other amplification systems to determine in one reaction the alleles of each locus contained within the multiplex system. [0001]
    • CITED REFERENCES
  • A full bibliographic citation of the references cited in this application can be found in the section preceding the claims. [0002]
    • DESCRIPTION OF THE PRIOR ART
  • In recent years, the discovery and development of polymorphic short tandem repeats (STRs) as genetic markers has stimulated progress in the development of linkage maps, the identification and characterization of diseased genes, and the simplification and precision of Genomic DNA typing. [0003]
  • Many loci, at least in the human genome, contain a polymorphic STR region. STR loci consist of short, repetitive sequence elements of 3 to 7 base pairs in length. It is estimated that there are 2,000,000 expected trimeric and tetrameric STRs present as frequently as once every 15 kilobases (kb) in the human genome (Edwards et al. 1991; Beckmann and Weber 1992). Nearly half of the STR loci studied by Edwards et al. (1991) are polymorphic, which provides a rich source of genetic markers. Variation in the number of repeat units at a particular locus is responsible for the observed polymorphism reminiscent of VNTR loci (Nakamura et al. 1987) and minisatellite loci (Jeffreys et al. 1985), which contain longer repeat units, and microsatellite or dinucleotide repeat loci (Litt and Luty 1989, Tautz 1989, Weber and May 1989, Beckmann and Weber 1992). [0004]
  • Polymorphic STR loci are extremely useful markers for human identification, paternity testing and genetic mapping. STR loci may be amplified via the polymerase chain reaction (PCR) by employing specific primer sequences identified in the regions flanking the tandem repeat. [0005]
  • Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another following electrophoretic separation by any suitable detection method including radioactivity, fluorescence, silver stain, and color. [0006]
  • To minimize labor, materials and analysis time, it is desirable to analyze multiple loci and/or more samples simultaneously. One approach for reaching this goal involves amplification of multiple loci simultaneously in a single reaction. Such “multiplex” amplifications have been described extensively in the literature. Multiplex amplification sets have been extensively developed for analysis of genes related to human genetic diseases such as Duchenne Muscular Dystrophy (Chamberlain et al. 1988, Chamberlain et al. 1989, Beggs et al. 1990, Clemens et al. 1991, Schwartz et al. 1992, Covone et al. 1992), Lesch-Nyhan Syndrome (Gibbs et al. 1990), Cystic Fibrosis (Estivill et al. 1991, Fortina et al. 1992, Ferrie et al. 1992, Morral and Estivill, 1992), and Retinoblasma (Lohmann et al. 1992). Multiplex amplification of polymorphic microsatellite markers (Clemens et al. 1991, Schwartz et al. 1992, Huang et al. 1992) and even STR markers (Edwards et al. 1992, Kimpton et al. 1993, Hammond et al. 1994) have been described. [0007]
  • These amplified products are generally separated by one of several methods of electrophoresis known to those skilled in the art. Several well-known methods of detection of the amplified products have also been described. While ethidium bromide staining of amplified fragments is employed in some cases, in others it is preferred to use methods which label only one of the two strands of the amplified material. Examples of this include radioactive or fluorescent labeling of one of the two primers prior to the amplification of a locus. One of the more sophisticated approaches to detection is the use of different fluorescent labels to allow detection of amplified materials representing different loci, but existing in the same space following electrophoresis. The products of the different loci are differentiated with the use of filters, which allow visualization of one fluorescent label at a time. [0008]
  • Reference is made to International Publications WO 93/18177 and WO 93/18178 to Fortina et al., which are directed to methods and kits for diagnosing diseases such as Cystic Fibrosis and β-thalassemia, respectively, using an allele-specific multiplex polymerase chain reaction system. According to Fortina et al., multiplex PCR has also been used for simultaneous amplification of multiple target sequences, permitting mutant allele scanning using two lanes of an agarose gel. [0009]
  • Ballabio et al. (1991), disclose a single tube multiplex allele specific PCR test using two different dye-tagged fluorescent primers for detection of the AF508 cystic fibrosis mutation. [0010]
  • While there are multiplex amplification procedures for specific loci, the use of multiplex amplification procedures is greatly desired for the detection of alleles in other types of loci such as specific STR loci. [0011]
    • SUMMARY OF THE INVENTION
  • It is therefore an object of the present invention to provide a method for the simultaneous amplification of multiple distinct polymorphic STR loci using PCR or other amplification systems to determine, in one reaction, the alleles of each locus contained within the multiplex. These combinations of specific loci into multiplexes have not been heretofore shown. [0012]
  • It is also an object of the present invention to provide a method and a kit specific for multiplex amplifications comprising specified loci. [0013]
  • These and other objects are addressed by the present invention which is directed to a method of simultaneously analyzing or determining the alleles present at each individual locus of each multiplex. This method comprises the steps of (1) obtaining at least one Genomic DNA sample to be analyzed, wherein the Genomic DNA sample has at least two loci which can be amplified together; (2) amplifying the STR sequences in the DNA sample; and (3) detecting the amplified materials in a fashion which reveals the polymorphic nature of the systems employed. [0014]
  • The present invention is also directed to a method of simultaneously analyzing multiple STR sequences wherein at least one of the loci is selected from the group consisting of: HUMCSFLPO, HUMTPOX, HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B), HUMLIPOL, HSAC04 (ACTBP2), HUMCYP19, HUMPLA2A1, HUMAPOA2, HUMCD4, HUMF13A01 and HUMMYOPK (Myotonic). [0015]
  • Specifically, the present invention is directed to a method of simultaneously analyzing multiple STR sequences in the following groups of loci: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX and HUMTH01; HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUMCYP19 and HUMPLA2A1; HUMCD4, HUMCSF1PO and HUMTH01; HUMCYP19, HUMFABP and HUMPLA2A1; HUMCYP19, HUMHPRTB and HUMPLA2A1; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMFESFPS and HUMLIPOL; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; HUMHPRTB, HUMBFXIII (F13B) and HUMTPOX; HUMHPRTB, HUMBFXIII (F13B) and HUMFESFPS; HUMCSF1PO, HUMTPOX and HUMCD4; HUMHPRTB, HUMFESFPS and HUMMYOPK (Myotonic); HUMCSFLPO, HUMTH01 and HUMCD4; HUMCSFLPO, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL; HUMCSFLPO, HUMTPOX, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and HUMCD4; and HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4. [0016]
  • The present invention provides a high throughput method for the detection and analysis of polymorphic genetic markers using specific combinations of loci and specified conditions. By selection of the appropriate detection method, the process can be used in laboratories which have only a power supply and a standard apparatus for/polyacrylamide gel electrophoresis or those which have the latest in equipment for fluorescent gel scanning, e.g., FluorImager™-575 (Molecular Dynamics, Sunnyvale, Calif.). Thus, the process of the present invention is adaptable for a variety of uses and laboratories. [0017]
  • The approach as specified in the present invention produces a savings in time, labor and materials in the analysis of loci contained within the multiplexes. The process of the present invention includes all the requisite primers, allowing between two and four or more loci to be amplified together in one amplification tube instead of amplifying each locus independently. [0018]
  • The present invention has specific use in the field of forensic analysis, paternity determination, monitoring of bone marrow transplantation, linkage mapping, and detection of genetic diseases and cancers. [0019]
  • These and other aspects of the present invention will become evident upon reference to the following detailed description of the invention and the attached drawings and photographs. [0020]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a photograph illustrating the simultaneous amplification of three loci: HUMCSF1PO, HUMTPOX and HUMTH01, with the amplified products of each locus shown migrating next to the corresponding allelic ladder for ease of interpretation in Example 1. [0021]
  • FIG. 2 is a computer image showing the fluorescent detection of multiplex amplification of the loci HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31 as detected with a FluorImager™-575 (Molecular Dynamics, Sunnyvale, Calif.) in Example 2. [0022]
  • FIG. 3 is a photograph showing the silver stain detection of the multiplex amplification in Example 3. [0023]
  • FIG. 4 is a computer image showing the fluorescent detection of multiplex amplification in Example 4. [0024]
  • FIG. 5 is a photograph showing the silver stain detection of the multiplex amplification in Example 5. [0025]
  • FIG. 6 is a photograph showing the silver stain detection of the multiplex amplification in Example 6. [0026]
  • FIG. 7 is a photograph showing the silver stain detection of the multiplex amplification in Example 7. [0027]
  • FIG. 8 is a photograph showing the silver stain detection of the multiplex amplification in Example 8. [0028]
  • FIG. 9 is a photograph showing the silver stain detection of the multiplex amplification in Example 9. [0029]
  • FIG. 10 is a photograph showing the silver stain detection of the multiplex amplification in Example 10. [0030]
  • FIG. 11 is a photograph showing the silver stain detection of the multiplex amplification in Example 11. [0031]
  • FIG. 12 is a photograph showing the silver stain detection of the multiplex amplification in Example 12. [0032]
  • FIG. 13 is a photograph showing the silver stain detection of the multiplex amplification in Example 13. [0033]
  • FIG. 14 is a photograph showing the silver stain detection of the multiplex amplification in Example 14. [0034]
  • FIG. 1S is a photograph showing the silver stain detection of the multiplex amplification in Example 15. [0035]
  • FIG. 16 is a photograph showing the silver stain detection of the multiplex amplification in Example 16. [0036]
  • FIG. 17 is a photograph showing the silver stain detection of the multiplex amplification in Example 17. [0037]
  • FIG. 18 is a photograph showing the silver stain detection of the multiplex amplification in Example 18. [0038]
  • FIG. 19 is a photograph showing the silver stain detection of the multiplex amplification in Example 19. [0039]
  • FIG. 20 is a photograph showing the silver stain detection of the multiplex amplification in Example 20. [0040]
  • FIG. 21 is a photograph showing the silver stain detection of the multiplex amplification in Example 21. [0041]
  • FIG. 22 is a photograph showing the silver stain detection of the multiplex amplification in Example 22. [0042]
  • FIG. 23 is a photograph showing the silver stain detection of the multiplex amplification in Example 23. [0043]
  • FIG. 25 is a photograph showing the silver stain detection of the multiplex amplification in Example 25. [0044]
  • FIG. 26 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 26. [0045]
  • FIG. 27 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 27. [0046]
  • FIG. 28 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 28. [0047]
  • FIG. 29 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 29. [0048]
  • FIG. 30 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 30. [0049]
  • FIG. 31 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 31. [0050]
  • FIG. 32 is a photograph of a computer image showing the fluorescent detection of the multiplex amplification in Example 32.[0051]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The following definitions are intended to assist in providing a clear and consistent understanding of the scope and detail of the terms: [0052]
  • Allelic ladder: a standard size marker consisting of amplified alleles from the locus. [0053]
  • Allele: a genetic variation/associated with a segment of Genomic DNA, i.e., one of two or more alternate forms of a DNA sequence occupying the same locus. [0054]
  • Biochemical nomenclature: standard biochemical nomenclature is used herein in which the nucleotide bases are designated as adenine (A); thymine (T); guanine (G); and cytosine (C). Corresponding nucleotides are, for example, deoxyguanosine-5′-triphosphate (dGTP). [0055]
  • Genomic DNA polymorphism: the condition in which two or more different nucleotide sequences coexist in the same interbreeding population in a Genomic DNA sequence. [0056]
  • Locus (or genetic locus): a specific position on a chromosome. Alleles of a locus are located at identical sites on homologous chromosomes. [0057]
  • Locus-specific primer: a primer that specifically hybridizes with a portion of the stated locus or its complementary strand, at least for one allele of the locus, and does not hybridize efficiently with other Genomic DNA sequences under the conditions used in the amplification method. [0058]
  • Polymerase chain reaction (PCR): a technique in which cycles of denaturation, annealing with primer, and extension with Genomic DNA polymerase are used to amplify the number of copies of a target Genomic DNA sequence by >10[0059] 16 times. The polymerase chain reaction process for amplifying nucleic acid is covered by U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference for a description of the process.
  • Polymorphism information content (PIC): a measure of the amount of polymorphism present at a locus (Botstein et al., 1980). PIC values range from 0 to 1.0, with higher values indicating greater degrees of polymorphism. This measure generally displays smaller values than the other commonly used measure, i.e., heterozygosity. For markers that are highly informative (heterozygosities exceeding about 70%), the difference between heterozygosity and PIC is slight. [0060]
  • Primary reaction: initial reaction using the purified human genomic DNA as template for the PCR. [0061]
  • Primers: two single-stranded oligonucleotides or DNA fragments which hybridize with opposing strands of a locus such that the 3′ termini of the primers are in closest proximity. [0062]
  • Primer pair: two [0063] primers including primer 1 that hybridizes to a single strand at one end of the Genomic DNA sequence to be amplified and primer 2 that hybridizes with the other end on the complementary strand of the Genomic DNA sequence to be amplified.
  • Primer site: the area of the target Genomic DNA to which a primer hybridizes. [0064]
  • Secondary reaction: reamplification with the same or different primer pair using a dilution of the primary reaction as template for the PCR. [0065]
  • Construction of the Multiplex System [0066]
  • Prior to constructing the multiplex system, an appropriate set of loci, primers, and amplification protocols must be selected such that amplification generates fragments such that alleles of the various loci do not overlap in size or, when such overlap occurs, fragments representing different loci are detectable by separate means. In addition, the selected loci must be compatible for use with a single amplification-protocol. The specific combinations of loci described herein are unique in this application. Combinations of loci may be rejected for either of these reasons, or because, in combination, one or more of the loci do not produce adequate product yield, or fragments which do not represent authentic alleles are produced in this reaction. [0067]
  • Successful combinations are generated by trial and error of locus combinations and by adjustment of primer concentrations to identify an equilibrium in which all included loci may be amplified. [0068]
  • Of particular importance in the multiplex system is the size range of amplified alleles produced from the individual loci which will be analyzed together. For ease of analysis with current technologies, systems which can be detected by amplification of fragments smaller than 500 bases were preferably selected. [0069]
  • The following multiplex combinations have been developed and are considered ideal combinations for use in the present system: [0070]
  • 1. HUMTH01 and HUMCSFLPO; [0071]
  • 2. HUMTH01 and HUMCD4; [0072]
  • 3. HUMTH01 and HUMTPOX; [0073]
  • 4. HUMF13A01 and HUMFABP; [0074]
  • 5. HUMF13A01 and HUMMYOPK (Myotonic); [0075]
  • 6. HUMF13A01 and HUMBFXIII (F13B); [0076]
  • 7. HUMBFXIII (F13B) and HUMFESFPS; [0077]
  • 8. HUMBFXIII (F13B) and HUMLIPOL; [0078]
  • 9. HUMHPRTB and HUMFESFPS; [0079]
  • 10. HSAC04 (ACTBP2) and HUMCYP19; [0080]
  • 11. HSAC04 (ACTBP2) and HUMFABP; [0081]
  • 12. HUMCSFLPO, HUMTPOX and HUMTH01; [0082]
  • 13. HUMHPRTB, HUMFESFPS and HUMVWFA31; [0083]
  • 14. HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1; [0084]
  • 15. HUMAPOA2, HUMCYP19 and HUMPLA2A1; [0085]
  • 16. HUMCD4, HUMCSF1PO and HUMTH01; [0086]
  • 17. HUMCYP19, HUMFABP and HUMPLA2A1; [0087]
  • 18. HUMCYP19, HUMHPRTB and HUMPLA2A1; [0088]
  • 19. HUMF13A01, HUMFABP and HUMCD4; [0089]
  • 20. HUMHPRTB, HUMFESFPS and HUMLIPOL; [0090]
  • 21. HUMF13A01, HUMFABP and HUMCD4; [0091]
  • 22. HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; [0092]
  • 23. HUMHPRTB, HUMBFXIII (F13B) and HUMTPOX; [0093]
  • 24. HUMHPRTB, HUMBFXIII (F13B) and HUMFESFPS; [0094]
  • 25. HUMCSFLPO, HUMTPOX and HUMCD4; [0095]
  • 26. HUMHPRTB, HUMFESFPS and HUMMYOPK (Myotonic); [0096]
  • 27. HUMCSF1PO, HUMTH01 and HUMCD4; [0097]
  • 28. HUMCSF1PO, HUMTH01 and HUMVWFA31; [0098]
  • 29. HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL; [0099]
  • 30. HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31; [0100]
  • 31. HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL; [0101]
  • 32. HUMCSF1PO, HUMTPOX, HUMTH01 and HUMCD4; and [0102]
  • 33. HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4. [0103]
  • The primers must also be designed so that the size of the resulting amplification products differ in length, thereby facilitating assignment of alleles to individual loci during detection. Inappropriate selection of primers can produce several undesirable effects such as lack of amplification, amplification at multiple sites, primer dimer formation, undesirable interaction of primer sequences from different loci, production of alleles from one locus which overlap with alleles from another, or requirement for amplification conditions or protocols for the different loci which are incompatible in a multiplex. The synthesis of the primers is conducted by procedures known to those skilled in the art. [0104]
  • Using Multiplexes of Two Loci to Develop Multiplexes Using More than Two Loci [0105]
  • Once a multiplex containing two loci is developed, it may be used as a core to create multiplexes containing more than two loci. New combinations are created including the first two loci. For example, the core multiplex containing loci HUMTH01 and HUMCSF1PO was used to generate derivative multiplexes of HUMTH01, HUMCSFLPO, and HUMTPOX; HUMTH01, HUMCSF1PO, and HUMCD4; HUMTH01, HUMCSF1PO, and HUMVWFA31; HUMTH01, HUMCSF1PO, HUMVWFA31, and HUMTPOX; and HUMTH01, HUMCSFLPO, HUMCD4, and HUMTPOX. Many other derivative multiplexes can be generated based upon a working multiplex. The derivative multiplexes are, in some sense, routine extensions of the core multiplex. [0106]
  • Preparation of Genomic DNA [0107]
  • All methods of Genomic DNA preparation which are compatible with the amplification process for a single locus should be appropriate for multiplex amplification. Many examples of preparation methods have been described in the literature (Patel et al. 1984, Gill et al. 1985). Genomic DNA concentrations are measured fluorometrically (Brunk et al. 1979). [0108]
  • Amplification of Genomic DNA [0109]
  • Human genomic DNA samples are subjected to PCR amplification using primers and thermocycling conditions specific for each locus. Reference is made to Table 1 for details of the primer sequences. The amplification protocol specific to each multiplex is listed in the specific examples. [0110]
    Sequence
    Designation Primer sequences ID Number
    HSAC04 primer 1: ACA TCT CCC CTA CCG CTA TA 1
    (ACTBP2) primer 2: AAT CTG GGC GAC AAG AGT GA 2
    HUMAPOA2 primer 1: GGA GCA GTC CTA GGG CCG CGC CGT 3
    (APOCIII) primer 2: GTG ACA GAG GGA GAC TCC ATT AAA 4
    HUMCSF1PO primer 1: AAC CTG AGT CTG CCA AGG ACT AGC 5
    primer 2: TTC CAC ACA CCA CTG GCC ATC TTC 6
    HUMCYP19 primer 1: GCA GGT ACT TAG TTA GCT AC 7
    (CYARP450) primer 2: TTA CAG TGA GCC AAG GTC GT 8
    HUMCD4 primer 1: CCA GGA AGT TGA GGC TGC AGT GAA 9
    primer 2: TTG GAG TCG CAA GCT GAA CTA GCG 10
    HUMF13A01 primer 1: GAG GTT GCA CTC CAG CCT TTG CAA 11
    primer 2: TTC CTG AAT CAT CCC AGA GCC ACA 12
    HUMBFXIII primer 1: TGA GGT GGT GTA CTA CCA TA 13
    (F13B) primer 2: GAT CAT GCC ATT GCA CTC TA 14
    HUMFABP primer 1: GTA GTA TCA GTT TCA TAG GGT CAC C 15
    primer 2: CAG TTC GTT TCC ATT GTC TGT CCG 16
    HUMFESFPS primer 1: GCT GTT AAT TCA TGT AGG GAA GGC 17
    primer 2: GTA GTC CCA GCT ACT TGG CTA CTC 18
    HUMHPRTB primer 1: ATG CCA CAG ATA ATA CAC ATC CCC 19
    (HPRT-1) primer 2: CTC TCC AGA ATA GTT AGA TGT AGG 20
    HUMNYOPK primer 1: GCT CGA AGG GTC CTT GTA GCC GGG 21
    Myotonic primer 2: GAT AGG TGG GGG TGC GTG GAG GAT 22
    HUMLIPOL primer 1: CTG ACC AAG GAT AGT GGG ATA TAG 23
    primer 2: GGT AAC TGA GCG AGA CTC TGT CT 24
    HUMPLA2A1 primer 1: GGT TGT AAG CTC CAT GAG GTT AGA 25
    (PLA-AZ) primer 2: TTG AGC ACT TAC TAT GTG CCA GGC T 26
    HUMTH01 primer 1: GTG GGC TGA AAA GCT CCC GAT TAT 27
    primer 2: ATT CAA AGG GTA TCT GGG CTC TGG 28
    HUMTPOX primer 1: ACT GGC ACA GAA CAG GCA CTT AGG 29
    primer 2: GGA GGA ACT GGG AAC CAC ACA GGT 30
    HUMVWFA31 primer 1: GA AAG CCC TAG TGG ATG AGA ATA ATC 31
    primer 2: GGA CAG ATG ATA AAT ACA TAG GAT GGA TGG 32
  • Reference is made to the examples below for additional details of the specific procedure relating to each multiplex. The locus-specific primers include a number of nucleotides which, under the conditions used in the hybridization, are sufficient to hybridize with an allele of the locus to be amplified and to be essentially free from amplification of alleles of other loci. Reference is made to U.S. Pat. No. 5,192,659 to Simons, which is incorporated herein by reference for a more detailed description of locus-specific primers. [0111]
  • Separation and Detection of Genomic DNA Fragments [0112]
  • Following amplification, products are then separated by electrophoresis, e.g., denaturing polyacrylamide gel electrophoresis (Sambrook et al., 1989). Preferred gel preparation and electrophoresis procedures are conducted as described in Example 1. Fragment separation occurs based on size and charge of the sample. [0113]
  • The Genomic DNA is then detected by, e.g., silver staining (Bassam et al. 1991). Alternatively, if radioactively-labeled or fluorescently-labeled primers were used for each locus, the products are detected by means available to detect these reporters as known to those skilled in the art. Amplified materials may be detected using any of a number of reporters including, e.g., silver staining, radioisotopes, fluorescers, chemiluminescers and enzymes in combination with detectable substrates. [0114]
  • Individual Genomic DNA samples containing amplified alleles are preferably compared with a size standard such as a Genomic DNA marker or locus-specific allelic ladder to determine the alleles present at each locus within the sample. The preferred size marker for evaluation of a multiplex amplification containing two or more polymorphic STR loci consists of a combination of allelic ladders for the loci being evaluated. [0115]
  • The preferred size marker for evaluation of a multiplex amplification containing two or more polymorphic STR loci which are generated using fluorescently-labeled primers for each locus consists of a combination of fluorescently-labeled allelic ladders for the loci being evaluated. [0116]
  • Following the construction of allelic ladders for individual loci, they may be mixed and loaded for gel electrophoresis at the same time as the loading of amplified samples occurs. Each allelic ladder co-migrates with alleles in the sample from the corresponding locus. [0117]
  • A permanent record of the data can be generated with the use of electrophoresis duplicating film (STR systems manual #TMD004, Promega Corporation, Madison, Wis.). [0118]
  • Advantage of Fluorescent Detection [0119]
  • With the advent of automated fluorescent imaging, faster detection and analysis of multiplex amplification products can be achieved. For fluorescent analyses, one fluoresceinated primer can be included in the amplification of each locus. Separation of the amplified fragments is achieved in precisely the same manner as with the silver stain detection method. The resulting gel is loaded onto a FluorImager® 575 (Molecular Dynamics, Sunnyvale, Calif.) which scans the gel and digitizes the data in three minutes. The FluorImager® contains an argon laser emitting 488 nm light which sweeps through the gel using a galvanometer-controlled mirror. The light activates fluorescent molecules in its path and they, in turn, emit light of higher wavelength. A filter prohibits passage of the original light, but allows collection of the emitted light by a fiber optic collector. A second filter selected by the user may be inserted between the fiber optic collector and the photomultiplier, allowing detection of specific wavelength bands (or colors) with each scan. [0120]
  • The image has an overall cleaner appearance than that obtained with the silver stain for three reasons. First, only one of the two PCR product strands is labeled with primer, simplifying the two band per allele images of the silver stain. Second, in the silver stain reaction, the entire gel is exposed to silver and prone to silver deposition causing a significant general background. With the fluorescent reporter, only the primer is labeled and the unincorporated primers migrate out of the bottom of the gel prior to detection. Third, some artifact bands of the PCR reaction are plentiful, but contain very little primer. [0121]
  • Because this fluorescent method detects only products with one particular primer, some of these artifacts which appear in silver stain of multiplex amplifications are not detected. In fact, this characteristic has allowed development of the more complex quadriplex as shown in FIG. 2 in place of the triplex shown in FIG. 1. [0122]
    • Kit
  • The present invention is also directed to kits that utilize the process described. A basic kit includes a container having a locus-specific primer pair (or alternately separate containers containing each primer of a primer pair) for each locus. The kit also includes instructions for use. [0123]
  • Other ingredients may include an allelic ladder directed to each of the specified loci, a sufficient quantity of enzyme for amplification, amplification buffer to facilitate the amplification, loading solution for preparation of the amplified material for gel electrophoresis, human genomic DNA as a control to test that the system is working well, a size marker to insure that materials migrate as anticipated in the gel, and a protocol and manual to educate the user and to limit error in use. The amounts of the various reagents in the kits can be varied depending upon a number of factors, such as the optimum sensitivity of the process. The instructions for use are suitable to enable any analyst to carry out the desired test. It is within the scope of this invention to provide test kits for use in manual applications or test kits for use with automated detectors or analyzers. [0124]
    • EXAMPLES
  • The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure or protection granted by the patent. [0125]
  • Genomic DNA isolation and quantitation were performed essentially as described by Puers et al., 1993. These methods are generally known to those skilled in the art and are preferred, but not required, for application of the invention. [0126]
  • Amplification products were separated by electrophoresis through a 0.4 mm thick 4% denaturing polyacrylamide gel (19:1 ratio of acrylamide to bis-acrylamide) which contained 7 M urea (Sambrook et al., 1989) and was chemically cross-linked to one glass plate (Kobayashi, 1988). Genomic DNA samples were mixed with 3 μl loading solution (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol), denatured at 95° C. for 2 min., and chilled on ice prior to loading. [0127]
  • Electrophoresis was performed at 60 W in 0.5×TBE for 1-2 hrs. The Genomic DNA was detected by silver staining (Bassam et al., 1991). Permanent images were obtained by exposure to Electrophoresis Duplicating Films (EDF, Kodak, Cat. No. 809 6232). Alternatively, detection can be performed by fluorescent scanning (Schumm et al., 1994) or radioactive detection (Hammond et al., 1994). [0128]
    • EXAMPLE 1 Silver Stain Detection of Multiplex Amplification of Loci HUMCSFLPO, HUMTPOX, and HUMTH01
  • In this example, a Genomic DNA template (three Genomic DNA samples) was amplified at the individual loci HUMCSFLPO, HUMTPOX, and HUMTH01 simultaneously in a single reaction vessel. The PCR amplifications were performed in 50 μl volumes using 25 ng template, 0.03 U Taq Genomic DNA Polymerase/[0129] μl 1×STR Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100, 1.5 mM MgCl2 and 200 μM each of dATP, dCTP, dGTP and dTTP), and using a Thermal Cycler 480 (Perkin Elmer Cetus). Amplification protocol 1 (96° C. for 2 min., then 10 cycles of 94° C. for 1 min., 64° C. for 1 min., and 70° C. for 1.5 min., followed by 20 cycles of 90° C. for 1 min., 64° C. for 1 min., 70° C. for 1.5 min.) was employed.
  • Six amplification primers were used in combination, including 0.2 μM each HUMCSF1PO primers 1 [SEQ. ID. 5] and 2 [SEQ. ID. 6], 0.2 μM each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 0.6 μM each HUMTH01 primers 1 [SEQ. ID. 27] and 2 [SEQ. ID. 28]. [0130]
  • Amplified products were separated by denaturing acrylamide gel electrophoresis on a 40 cm gel for 60-90 min. at 60 W and products were visualized by silver stain analysis according the protocol of Bassam et al. (1991). [0131]
  • Reference is made to FIG. 1 which reveals the silver stain detection of the multiplex amplification. [0132] Lanes 2, 3, and 5 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSFLPO, HUMTPOX, and HUMTH01. Lanes 1, 4, and 7 contain allelic ladders for the three loci and lane 6 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 2 Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMVWFA31
  • In this example, a Genomic DNA template was amplified at the individual loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMVWFA31 simultaneously in a single reaction vessel. The PCR amplifications were performed in 25 μl volumes using 25 ng template, 0.04 U Taq Genomic DNA Polymerase/μl, 1×STR Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100, 1.5 mM MgCl2 and 200 μM each of DATP, dCTP, dGTP and dTTP), and using a Thermal Cycler 480 (Perkin Elmer Cetus). [0133] Amplification protocol 1, as described in Example 1, was employed. Eight amplification primers were used in combination, including 1 μM each HUMCSF1PO primer 2 [SEQ. ID. 5] and fluorescein-labeled primer 1 [SEQ. ID. 5], 0.15 μM each HUMTPOX primer 1 [SEQ. ID. 29] and fluorescein-labeled primer 2 [SEQ. ID. 30], 0.2 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 271, and 1M each HUMVWFA31 primer 1 [SEQ. ID. 31] and fluorescein-labeled primer 2 [SEQ. ID. 32].
  • Amplified products were separated by denaturing acrylamide gel electrophoresis on a 32 cm gel for 45 minutes at 40 watts. Detection of the fluorescent signal was achieved using the FluorImager™575 (Molecular Dynamics, Sunnyvale, Calif.). Reference is made to FIG. 2 which is a computer image of a FluorImager scan. Lanes 2-7 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMVWFA31. [0134] Lane 1 contains allelic ladders for the 4 loci.
    • EXAMPLE 3 Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and HUMVWFA31
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMVWFA31 simultaneously in a single reaction vessel. The PCR amplifications were performed in 25 μl volumes using 25 ng template, 0.03 U Taq Genomic DNA Polymerase/μl, 1×STR Buffer (described in example 1), and a Thermal Cycler 480 (Perkin Elmer Cetus). Amplification protocol 2 (96° C. for 2 min., then 10 cycles of 94° C. for 1 min., 60° C. for 1 min., and 70° C. for 1.5 min., followed by 20 cycles of 90° C. for 1 min., 64° C. for 1 min., 70° C. for 1.5 min.) was employed. Amplified products were separated by denaturing acrylamide gel electrophoresis on a 32 cm gel for 45 min. at 40 W and products were visualized by silver stain analysis according the protocol of Bassam et al. (supra.). Six primers were used in combination including 0.2 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], 1.5 μM each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 1 μM each HUMVWFA31 primers 1 [SEQ. ID. 31] and 2 [SEQ. ID. 32]. [0135]
  • Reference is made to FIG. 3 which reveals the silver stain detection of the multiplex amplification. Lanes 2-6 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMVWFA31. [0136] Lanes 1 and 7 contain allelic ladders for the 3 loci.
    • EXAMPLE 4 Fluorescent Detection of Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS. HUMBFXIII (F13B), and HUMLIPOL
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B), and HUMLIPOL simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 2 using [0137] amplification protocol 2, as described in Example 3.
  • Eight primers were used in combination, including 1M each HUMHPRTB primer 2 [SEQ. ID. 20] and fluorescein-labeled primer 1 [SEQ. ID. 19], 2.5 μM each HUMFESFPS primer 2 [SEQ. ID. 18] and fluorescein-labeled primer 1 [SEQ. ID. 17], 1 μM each HUMBFXIII (F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1 [SEQ. ID. 13], and 0.5 μM each HUMLIPOL primer 2 [SEQ. ID. 24] and fluorescein-labeled primer 1 [SEQ. ID. 23]. [0138]
  • Reference is made to FIG. 4 which is a computer image of a FluorImager scan. Lanes 2-7 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B), and HUMLIPOL. [0139] Lane 1 contains allelic ladders for the 4 loci.
    • EXAMPLE 5 Multiplex Amplification of Loci HSAC04 (ACTBP2) and HUMCYP19
  • In this example, a Genomic DNA template was amplified at the individual loci HSAC04 and HUMCYP19 simultaneously in a single reaction vessel. The PCR amplifications were performed in 15 μl volumes with 25 ng template, 0.01 U Taq Genomic DNA Polymerase/μl, 1×Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 μM each of dATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus). [0140] Amplification protocol 2, as described in Example 3, was employed. Amplified products were separated and detected per example 1. Four primers were used in combination, including 1 μM each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], and 1 μM each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8].
  • Reference is made to FIG. 5 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2) and HUMCYP19. [0141] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 6 Multiplex Amplification of Loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1
  • In this example, a Genomic DNA template was amplified at the loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1 simultaneously in a single reaction vessel. The PCR amplifications were performed in 15 μl volumes with 25 ng template, 0.02 U Taq DNA Polymerase/μl, 1×Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 μM each of DATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus). [0142] Amplification protocol 2, as described in Example 3, was employed. Amplified products were separated and detected per example 1. Six primers were used in combination, including 1 μM each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], 1 μM each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26], and 1 μM each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8].
  • Reference is made to FIG. 6 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2), HUMCYP19, and HUMPLA2A1. [0143] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 7
  • Multiplex Amplification of Loci HSAC04 (ACTBP2) and HUMFABP [0144]
  • In this example, a Genomic DNA template was amplified at the loci HSAC04 (ACTBP2) and HUMFABP simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 5 using [0145] amplification protocol 2, as described in Example 3. Four primers were used in combination, 1 μM each HSAC04 (ACTBP2) primers 1 [SEQ. ID. 1] and 2 [SEQ. ID. 2], and 1 μM each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16].
  • Reference is made to FIG. 7 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HSAC04 (ACTBP2) and HUMFABP. [0146] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 8 Multiplex Amplification of Loci HUMAPOA2, HUMCYP19, and HUMPLA2A1
  • In this example, a Genomic DNA template was amplified at the loci HUMAPOA2, HUMCYP19, and HUMPLA2A simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 6 using [0147] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMAPOA2 primers 1 [SEQ. ID. 3] and 2 [SEQ. ID. 4], 1 μM each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], and 1M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26]. Reference is made to FIG. 8 which reveals the silver stain detection of the multiplex amplification. Lanes 1 and 3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMAPOA2, HUMCYP19, and HUMPLA2A1. Lane 2 contains a Genomic DNA sample which failed to amplify and lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 9 Multiplex Amplification of Loci HUMCD4. HUMCSFLPO, and HUMTH01
  • In this example, a Genomic DNA template was amplified at the loci HUMCD4, HUMCSF1PO, and HUMTH01 simultaneously in a single reaction vessel. The PCR amplifications were performed in 50 μl volumes with 25 ng template, 0.02 U Taq Genomic DNA Polymerase/μl, 1×Taq Genomic DNA Polymerase Buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 0.1% Triton X-100 and 1.5 mM MgCl2) and 200 μM each of DATP, dCTP, dGTP and dTTP using a Thermal Cycler 480 (Perkin Elmer Cetus). [0148] Amplification protocol 1, as described in Example 1, was employed. Amplified products were separated and detected as described in Example 1. Six primers were used in combination, including 1 μM each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10], 1 μM each HUMCSF1PO primers 1 [SEQ. ID. 5] and 2 [SEQ. ID. 6], and 1 μM each HUMTH01 primers 1 [SEQ. ID. 27] and 2 [SEQ. ID. 28].
  • Reference is made to FIG. 9 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCD4, HUMCSF1PO, and HUMTH01. [0149] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 10 Multiplex Amplification of Loci HUMCYP19. HUMFABP, and HUMPLA2A1
  • In this example, a Genomic DNA template was amplified at the loci HUMCYP19, HUMFABP, and HUMPLA2A1 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 6 using [0150] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], 1 μM each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16] and 1 μM each HUMPLA1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Reference is made to FIG. 10 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCYP19, HUMFABP, and HUMPLA2A1. [0151] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 11 Multiplex Amplification of Loci HUMCYP19. HUMHPRTB, and HUMPLA2A1
  • In this example, a Genomic DNA template was amplified at the loci HUMCYP19, HUMHPRTB, and HUMPLA2A1 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 9 using [0152] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMCYP19 primers 1 [SEQ. ID. 7] and 2 [SEQ. ID. 8], 1 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], and 1 μM each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Reference is made to FIG. 11 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCYP19, HUMHPRTB, and HUMPLA2A1. [0153] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 12 Multiplex Amplification of Loci HUMF13A01 and HUMFABP
  • In this example, a Genomic DNA template was amplified at the loci HUMF13A01 and HUMFABP simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 5 using [0154] amplification protocol 1, as described in Example 1. Four primers were used in combination, including 1 μM each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12], and 1 μM each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16].
  • Reference is made to FIG. 12 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMFABP. [0155] Lane 4 displays a sample without DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 13 Multiplex Amplification of Loci HUMBFXIII (F13B) and HUMFESFPS
  • In this example, a Genomic DNA template was amplified at the loci HUMBFXIII (F13B) and HUMFESFPS simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 6 using [0156] amplification protocol 1, as described in Example 1. Four primers were used in combination, including 1 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14], and 1 μM each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18].
  • Reference is made to FIG. 13 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B) and HUMFESFPS. [0157] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 14 Multiplex Amplification of Loci HUMBFXIII (F13B). HUMHPRTB, and HUMPLA2A1
  • In this example, a Genomic DNA template was amplified at the loci HUMBFXIII (F13B), HUMHPRTB, and HUMPLA2A1 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 6 using [0158] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14], 1M each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], and 1M each HUMPLA2A1 primers 1 [SEQ. ID. 25] and 2 [SEQ. ID. 26].
  • Reference is made to FIG. 14 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B), HUMHPRTB, and HUMPLA2A1. [0159] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 15 Multiplex Amplification of Loci HUMF13A01, HUMFABP, and HUMCD4
  • In this example, a Genomic DNA template was amplified at the loci HUMF13A01, HUMFABP, and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 5 using [0160] amplification protocol 1, as described in Example 1. Six primers were used in combination, including 1 μM each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12], 1M each HUMFABP primers 1 [SEQ. ID. 15] and 2 [SEQ. ID. 16], and 1M each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 101.
  • Reference is made to FIG. 15 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01, HUMFABP, and HUMCD4. [0161] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 16 Multiplex Amplification of Loci HUMHPRTB and HUMFESFPS
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB and HUMFESFPS simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using 500-0.5 ng template, 0.02 U Taq Genomic DNA Polymerase/A1 and [0162] amplification protocol 2, as described in Example 3. Four primers were used in combination, including 0.2 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20] and 1.5 μM each HUMFESFPS primers 1 [SEQ. ID. 171 and 2 [SEQ. ID.
  • 18). [0163]
  • Reference is made to FIG. 16 which reveals the silver stain detection of the multiplex amplification. Lanes 1-6 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB and HUMFESFPS-using 500, 50, 25, 5, 1 and 0.5 ng DNA template. [0164] Lane 7 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 17 Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and HUMLIPOL
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMLIPOL simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using [0165] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 0.4 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], 3 μM each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 2 μM each HUMLIPOL primers 1 [SEQ. ID. 23] and 2 [SEQ. ID. 24].
  • Reference is made to FIG. 17 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS and HUMLIPOL. [0166] Lane 4 displays a sample without Genomic DNA subjected to the same procedures, i.e., a negative control.
    • EXAMPLE 18 Multiplex Amplification of Loci HUMBFXIII (F13B) and HUMLIPOL
  • In this example, a Genomic DNA template was amplified at the loci HUMBFXIII (F13B) and HUMLIPOL Simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/pl and [0167] amplification protocol 2, as described in Example 3. Four primers were used in combination, including 1 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14] and 1 μM each HUMLIPOL primers 1 [SEQ. ID. 23] and 2 [SEQ. ID. 24].
  • Reference is made to FIG. 18 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMBFXIII (F13B) and HUMLIPOL. [0168]
    • EXAMPLE 19 Multiplex Amplification of Loci HUMHPRTB. HUMTPOX, and HUMBFXIII (F13B)
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMTPOX, and HUMBFXIII (F13B) simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using [0169] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1M each HUMHPRTB primers 1. [SEQ. ID. 19] and 2 [SEQ. ID. 20], 0.2 μM each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 2 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Reference is made to FIG. 19 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMTPOX, and HUMBFXIII (F13B). [0170]
    • EXAMPLE 20 Multiplex Amplification of Loci HUMHPRTB. HUMFESFPS, and HUMBFXIII (F13B)
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMBFXIII (F13B) simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using [0171] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 [SEQ. ID. 20], 2 μM each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 2 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Reference is made to FIG. 20 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMBFXIII (F13B). [0172]
    • EXAMPLE 21 Multiplex Amplification of Loci HUMCSFLPO. HUMTPOX, and HUMCD4
  • In this example, a Genomic DNA template was amplified at the loci HUMCSF1PO, HUMTPOX, and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using [0173] amplification protocol 1, as described in Example 1. Six primers were used in combination, including 1 μM each HUMCSFLPO primers 1 [SEQ. ID. 5] and 2 (SEQ. ID. 61], 1 μM each HUMTPOX primers 1 [SEQ. ID. 29] and 2 [SEQ. ID. 30], and 1 μM each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10].
  • Reference is made to FIG. 21 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, and HUMCD4. [0174]
    • EXAMPLE 22 Multiplex Amplification of Loci HUMHPRTB, HUMFESFPS, and HUMMYOPK (Myotonic)
  • In this example, a Genomic DNA template was amplified at the loci HUMHPRTB, HUMFESFPS, and HUMMYOPK simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using [0175] amplification protocol 2, as described in Example 3. Six primers were used in combination, including 1 μM each HUMHPRTB primers 1 [SEQ. ID. 19] and 2 (SEQ. ID. 20], 1M each HUMFESFPS primers 1 [SEQ. ID. 17] and 2 [SEQ. ID. 18], and 1 μM each HUMMYOPK (Myotonic) primers 1 [SEQ. ID. 21] and 2 [SEQ. ID. 22].
  • Reference is made to FIG. 22 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMFESFPS, and HUMMYOPK (Myotonic). [0176]
    • EXAMPLE 23 Multiplex Amplification of Loci HUMCSF1PO. HUMTPOX. HUMTH01, and HUMCD4
  • In this example, a Genomic DNA template was amplified at the loci HUMCSFLPO, HUMTPOX, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and [0177] amplification protocol 1, as described in Example 1. Eight primers were, used in combination, including 1 μM each HUMCSF1PO primers 1 (SEQ. ID. 5] and 2 (SEQ. ID. 6], 1 μM each HUMTPOX primers 1 [SEQ. ID. 291 and 2 [SEQ. ID. 30], 1 μM each HUMTH01 primers 1 (SEQ. ID. 27] and 2 [SEQ. ID. 28], and 1 μM each HUMCD4 primers 1 [SEQ. ID. 9] and 2 [SEQ. ID. 10].
  • Reference is made to FIG. 23 which reveals the silver stain detection of the multiplex amplification. [0178]
  • Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4. [0179]
    • EXAMPLE 24 Multiplex Amplification of Loci HUMF13A01 and HUMMYOPK (Myotonic)
  • In this example, a Genomic DNA template was amplified at the loci HUMF13A01 and HUMMYOPK (Myotonic) simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and [0180] amplification protocol 1, as described in Example 1. Four primers were used in combination, including 0.1 μM each HUMF13A01 primers 1 [SEQ. ID. 11] and 2 [SEQ. ID. 12] and 1 μM each HUMMYOPK (Myotonic) primers 1 [SEQ. ID. 21] and 2 [SEQ. ID. 22].
  • Reference is made to FIG. 24 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMMYOPK (Myotonic). [0181]
    • EXAMPLE 25 Multiplex Amplification of Loci HUMF13A01 and HUMBFXIII (F13B)
  • In this example, a Genomic DNA template was amplified at the loci HUMF13A01 and HUMBFXIII (F13B) simultaneously in a single reaction vessel. The PCR amplifications and other manipulations were performed as described in Example 1 using 0.03 U Taq Genomic DNA Polymerase/A1 and [0182] amplification protocol 2, as described in Example 3. Four primers were used in combination, including 0.1 μM each HUMF13A01 primers 1 [SEQ. ID. 11] and 2-[SEQ. ID. 12] and 0.5 μM each HUMBFXIII (F13B) primers 1 [SEQ. ID. 13] and 2 [SEQ. ID. 14].
  • Reference is made to FIG. 25 which reveals the silver stain detection of the multiplex amplification. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMF13A01 and HUMBFXIII (F13B). [0183]
    • EXAMPLE 26 Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO. HUMTPOX. HUMTH01, and HUMCD4
  • In this example, a Genomic DNA template was amplified at the individual loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.04 U Taq Genomic DNA Polymerase/A1 and [0184] amplification protocol 1, as described in Example 1. Eight amplification primers were used in combination, including 2 μM each HUMCSFLPO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 0.5 μM each HUMTPOX primer 1 [SEQ. ID. 29] and fluorescein-labeled primer 2 [SEQ. ID. 30], 0.5 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27] and 0.5 μM each HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID. 10].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 26 which is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTPOX, HUMTH01, and HUMCD4. [0185]
    • EXAMPLE 27 Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO. HUMTH01, and HUMCD4
  • In this example, a Genomic DNA template was amplified at the individual loci HUMCSF1PO, HUMTH01, and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and [0186] amplification protocol 1, as described in Example 1. Six amplification primers were used in combination, including 1 μM each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 1 μM each HUMTH01 primer 2 [SEQ. ID. 281 and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 μM each HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID. 10].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 27 which is a photograph of a computer image of a FluorImager scan. [0187] Lanes 1 and 2 contain DNA samples simultaneously co-amplified for the loci HUMCSFLPO, HUMTH01, and HUMCD4.
    • EXAMPLE 28 Fluorescent Detection of Multiplex Amplification of Loci HUMCSF1PO. HUMTH01, and HUMVWFA31
  • In this example, a Genomic DNA template was amplified at the individual loci HUMCSFLPO, HUMTH01, and HUMVWFA31 simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and [0188] amplification protocol 1, as described in Example 1. Six amplification primers were used in combination, including 1M each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5], 1 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27], and 1 μM each HUMVWFA31 primer 1 [SEQ. ID. 31] and fluorescein-labeled primer 2 [SEQ. ID. 32].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 28 which is a photograph of a computer image of a FluorImager scan. [0189] Lanes 1 and 2 contain DNA samples simultaneously co-amplified for the loci HUMCSF1PO, HUMTH01, and HUMVWFA31.
    • EXAMPLE 29 Fluorescent Detection of Multiplex Amplification of Loci HUMHPRTB. HUMBFXIII (F13B), and HUMLIPOL
  • In this example, a Genomic DNA template was amplified at the individual loci HUMHPRTB, HUMBFXIII (F13B), and HUMLIPOL simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.03 U Taq Genomic DNA Polymerase/A1 and [0190] amplification protocol 2, as described in Example 3. Six amplification primers were used in combination, including 1 μM each HUMHPRTB primer 2 (SEQ. ID. 20] and fluorescein-labeled primer 1 [SEQ. ID. 19], 1M each HUMBFXIII (F13B) primer 2 [SEQ. ID. 14] and fluorescein-labeled primer 1 [SEQ. ID. 13], and 1 μM each HUMLIPOL primer 2 [SEQ. ID. 24] and fluorescein-labeled primer 1 [SEQ. ID. 23].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 29 which is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMHPRTB, HUMBFXIII (F13B), and HUMLIPOL. [0191]
    • EXAMPLE 30 Fluorescent Detection of Multiplex Amplification of Loci HUMCSFLPO and HUMTH01
  • In this example, a Genomic DNA template was amplified at the individual loci HUMCSF1PO and HUMTH01 simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and [0192] amplification protocol 1, as described in Example 1. Four amplification primers were used in combination, including 2 μM each HUMCSF1PO primer 2 [SEQ. ID. 6] and fluorescein-labeled primer 1 [SEQ. ID. 5] and 1 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 30 which is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMCSFLPO and HUMTH01. [0193]
    • EXAMPLE 31 Fluorescent Detection of Multiplex Amplification of Loci HUMTH01 and HUMCD4
  • In this example, a Genomic DNA template was amplified at the individual loci HUMTH01 and HUMCD4 simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and [0194] amplification protocol 1, as described in Example 1. Four amplification primers were used in combination, including 1 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 μM each HUMCD4 primer 1 [SEQ. ID. 9] and fluorescein-labeled primer 2 [SEQ. ID. 10].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 31 which is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMTH01 and HUMCD4. [0195]
    • EXAMPLE 32 Fluorescent Detection of Multiplex Amplification of Loci HUMTH01 and HUMTPOX
  • In this example, a Genomic DNA template was amplified at the individual loci HUMTH01 and HUMTPOX simultaneously in a single reaction vessel. The PCR amplifications were performed as described in Example 1 using 0.02 U Taq Genomic DNA Polymerase/A1 and [0196] amplification protocol 1, as described in Example 1. Four amplification primers were used in combination, including 1 μM each HUMTH01 primer 2 [SEQ. ID. 28] and fluorescein-labeled primer 1 [SEQ. ID. 27] and 1 μM each HUMTPOX primer 2 [SEQ. ID. 30] and fluorescein-labeled primer 1 [SEQ. ID. 29].
  • Amplified products were detected as in Example 2. Reference is made to FIG. 32 which is a photograph of a computer image of a FluorImager scan. Lanes 1-3 contain Genomic DNA samples simultaneously co-amplified for the loci HUMTH01 and HUMTPOX. [0197]
  • It is understood that the invention is not confined to the particular construction and arrangements herein illustrated and described, but embraces such modified forms thereof and come within the scope of the claims following the bibliography. [0198]
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  • 1 32 20 base pairs nucleic acid single linear DNA (genomic) 1 ACATCTCCCC TACCGCTATA 20 20 base pairs nucleic acid single linear DNA (genomic) 2 AATCTGGGCG ACAAGAGTGA 20 24 base pairs nucleic acid single linear DNA (genomic) 3 GGAGCAGTCC TAGGGCCGCG CCGT 24 24 base pairs nucleic acid single linear DNA (genomic) 4 GTGACAGAGG GAGACTCCAT TAAA 24 24 base pairs nucleic acid single linear DNA (genomic) 5 AACCTGAGTC TGCCAAGGAC TAGC 24 24 base pairs nucleic acid single linear DNA (genomic) 6 TTCCACACAC CACTGGCCAT CTTC 24 20 base pairs nucleic acid single linear DNA (genomic) 7 GCAGGTACTT AGTTAGCTAC 20 20 base pairs nucleic acid single linear DNA (genomic) 8 TTACAGTGAG CCAAGGTCGT 20 24 base pairs nucleic acid single linear DNA (genomic) 9 CCAGGAAGTT GAGGCTGCAG TGAA 24 24 base pairs nucleic acid single linear DNA (genomic) 10 TTGGAGTCGC AAGCTGAACT AGCG 24 24 base pairs nucleic acid single linear DNA (genomic) 11 GAGGTTGCAC TCCAGCCTTT GCAA 24 24 base pairs nucleic acid single linear DNA (genomic) 12 TTCCTGAATC ATCCCAGAGC CACA 24 20 base pairs nucleic acid single linear DNA (genomic) 13 TGAGGTGGTG TACTACCATA 20 20 base pairs nucleic acid single linear DNA (genomic) 14 GATCATGCCA TTGCACTCTA 20 25 base pairs nucleic acid single linear DNA (genomic) 15 GTAGTATCAG TTTCATAGGG TCACC 25 24 base pairs nucleic acid single linear DNA (genomic) 16 CAGTTCGTTT CCATTGTCTG TCCG 24 24 base pairs nucleic acid single linear DNA (genomic) 17 GCTGTTAATT CATGTAGGGA AGGC 24 24 base pairs nucleic acid single linear DNA (genomic) 18 GTAGTCCCAG CTACTTGGCT ACTC 24 24 base pairs nucleic acid single linear DNA (genomic) 19 ATGCCACAGA TAATACACAT CCCC 24 24 base pairs nucleic acid single linear DNA (genomic) 20 CTCTCCAGAA TAGTTAGATG TAGG 24 24 base pairs nucleic acid single linear DNA (genomic) 21 GCTCGAAGGG TCCTTGTAGC CGGG 24 24 base pairs nucleic acid single linear DNA (genomic) 22 GATAGGTGGG GGTGCGTGGA GGAT 24 24 base pairs nucleic acid single linear DNA (genomic) 23 CTGACCAAGG ATAGTGGGAT ATAG 24 23 base pairs nucleic acid single linear DNA (genomic) 24 GGTAACTGAG CGAGACTGTG TCT 23 24 base pairs nucleic acid single linear DNA (genomic) 25 GGTTGTAAGC TCCATGAGGT TAGA 24 25 base pairs nucleic acid single linear DNA (genomic) 26 TTGAGCACTT ACTATGTGCC AGGCT 25 24 base pairs nucleic acid single linear DNA (genomic) 27 GTGGGCTGAA AAGCTCCCGA TTAT 24 24 base pairs nucleic acid single linear DNA (genomic) 28 ATTCAAAGGG TATCTGGGCT CTGG 24 24 base pairs nucleic acid single linear DNA (genomic) 29 ACTGGCACAG AACAGGCACT TAGG 24 24 base pairs nucleic acid single linear DNA (genomic) 30 GGAGGAACTG GGAACCACAC AGGT 24 29 base pairs nucleic acid single linear DNA (genomic) 31 GAAAGCCCTA GTGGATGATA AGAATAATC 29 30 base pairs nucleic acid single linear DNA (genomic) 32 GGACAGATGA TAAATACATA GGATGGATGG 30

Claims (20)

What is claimed is:
1. A method of simultaneously determining the alleles present in at least two loci from one or more Genomic DNA samples, comprising:
a. obtaining at least one Genomic DNA sample to be analyzed, wherein the Genomic DNA sample has at least two loci which can be amplified together;
b. amplifying the short tandem repeat sequences in the Genomic DNA sample; and
c. evaluating the amplified fragments to determine the alleles present at each amplified locus within the Genomic DNA sample.
2. The method of claim 1 wherein at least one of the loci is selected from the group consisting of: HUMCSFLPO, HUMTPOX, HUMVWFA31, HUMFESFPS, HUMBFXIII (F13B), HUMLIPOL, HSAC04 (ACTBP2), HUMCYP19, HUMAPOA2, HUMF13A01 and HUMMYOPK (Myotonic).
3. The method of claim 1 wherein at least two loci are selected from the groups consisting of: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; and HSAC04 (ACTBP2) and HUMFABP.
4. The method of claim 1 wherein the loci are selected from the group consisting of: HUMTH01 and HUMCSF1PO; HUMTH01 and HUMCD4; HUMTH01 and HUMTPOX; HUMF13A01 and HUMFABP; HUMF13A01 and HUMMYOPK (Myotonic); HUMF13A01 and HUMBFXIII (F13B); HUMBFXIII (F13B) and HUMFESFPS; HUMBFXIII (F13B) and HUMLIPOL; HUMHPRTB and HUMFESFPS; HSAC04 (ACTBP2) and HUMCYP19; HUMCSF1PO, HUMTPOX, and HUMTH01; HUMHPRTB, HUMFESFPS and HUMVWFA31; HSAC04 (ACTBP2), HUMCYP19 and HUMPLA2A1; HSAC04 (ACTBP2) and HUMFABP; HUMAPOA2, HUMCYP19 and HUMPLA2A1; HUMCD4, HUMCSF1PO and HUMTH01; HUMCYP-19, HUMFABP and HUMPLA1; HUMCYP19, HUMHPRTB and HUMPLA1; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMFESFPS and HUMLIPOL; HUMF13A01, HUMFABP and HUMCD4; HUMHPRTB, HUMBFXIII (F13B) and HUMPLA2A1; HUMHPRTB, HUMBFXIII (F13B) and HUMTPOX; HUMHPRTB, HUMBFXIII (F13B) and HUMFESFPS; HUMCSFLPO, HUMTPOX and HUMCD4; HUMHPRTB, HUMFESFPS and HUMMYOPK (Myotonic). HUMCSFLPO, HUMTH01 and HUMCD4; HUMCSF1PO, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31; HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL; HUMCSF1PO, HUMTPOX, HUMTH01 and HUMCD4; and HUMCSF1PO, HUMTH01, HUMTPOX and HUMCD4.
5. The method of claim 1 wherein the loci are HUMHPRTB and HUMFESFPS.
6. The method of claim 1 wherein the loci are HUMCSF1PO, HUMTPOX, and HUMTH01.
7. The method of claim 1 wherein the loci are HUMHPRTB, HUMFESFPS, HUMBFXIII (F13B) and HUMLIPOL.
8. The method of claim 1 wherein the loci are HUMCSF1PO, HUMTPOX, HUMTH01 and HUMVWFA31.
9. The method of claim 1 wherein the loci are HUMHPRTB, HUMFESFPS and HUMVWFA31.
10. The method of claim 1 wherein the Genomic DNA in step b. is amplified by polymerase chain reduction.
11. The method of claim 8 wherein the process of amplifying short tandem repeat sequences requires primer pairs selected from the group consisting of SEQ ID. NO. 1 and SEQ ID. NO. 2, SEQ ID. NO. 3 and SEQ ID. NO. 4, SEQ ID. NO. 5 and SEQ ID. NO. 6, SEQ ID. NO. 7 and SEQ ID. NO. 8, SEQ ID. NO. 9 and SEQ ID. NO. 10, SEQ ID. NO. 11 and SEQ ID. NO. 12, SEQ ID. NO. 13 and SEQ ID. NO. 14, SEQ ID. NO. 15 and SEQ ID. NO. 16, SEQ ID. NO. 17 and SEQ ID. NO. 18, SEQ ID. NO. 19 and SEQ ID. NO. 20, SEQ ID. NO. 21 and SEQ ID. NO. 22, SEQ ID. NO. 23 and SEQ ID. NO. 24, SEQ ID. NO. 25 and SEQ ID. NO. 26, SEQ ID. NO. 27 and SEQ ID. NO. 28, SEQ ID. NO. 29 and SEQ ID. NO. 30, and SEQ ID. NO. 31 and SEQ ID. NO. 32.
12. The method of claim 1 further comprising adding short tandem repeat allelic ladders containing nucleotide fragments of the same lengths as two or more known alleles for each of the loci and determining the allele content of the Genomic DNA sample by comparison with the amplified short tandem repeat fragments for each of the loci.
13. The method of claim 1 wherein the amplified short tandem repeat sequences are compared by polyacrylamide gel electrophoresis.
14. The method of claim 1 wherein the amplified short tandem repeat sequences are compared using silver stain analysis.
15. The method of claim 1 wherein the amplified short tandem repeat sequences are compared by fluorescent analysis.
16. The method of claim 1 further comprising identifying an appropriate set of loci and primers which provide non-overlapping alleles.
17. The method of claim 1 wherein the samples to be tested are selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine or other tissue, placental cells or fetal cells present in amniotic fluid and mixtures of body fluids.
18. A method of simultaneously determining the alleles present in at least two loci from one or more Genomic DNA samples, comprising:
a. identifying an appropriate set of loci and primers which provide non-overlapping alleles;
b. obtaining at least one Genomic DNA sample to be analyzed, wherein the Genomic DNA sample has at least two loci which can be amplified together;
c. amplifying the short tandem repeat sequences in the Genomic DNA sample; and
d. evaluating the amplified fragments to determine the alleles present at each amplified locus within the Genomic DNA sample.
19. A kit for simultaneously analyzing short tandem repeat sequences in at least two loci from one or more Genomic DNA samples, comprising:
a. a container containing oligonucleotide primer pairs for each of the specified loci; and
b. instructions for use.
20. The kit of claim 17 wherein the primer pairs are selected from the group of loci consisting of SEQ ID. NO. 1 and SEQ ID. NO. 2, SEQ ID. NO. 3 and SEQ ID. NO. 4, SEQ ID. NO. 5 and SEQ ID. NO. 6, SEQ ID. NO. 7 and SEQ ID. NO. 8, SEQ ID. NO. 9 and SEQ ID. NO. 10, SEQ ID. NO. 11 and SEQ ID. NO. 12, SEQ ID. NO. 13 and SEQ ID. NO. 14, SEQ ID. NO. 15 and SEQ ID. NO. 16, SEQ ID. NO. 17 and SEQ ID. NO. 18, SEQ ID. NO. 19 and SEQ ID. NO. 20, SEQ ID. NO. 21 and SEQ ID. NO. 22, SEQ ID. NO. 23 and SEQ ID. NO. 24, SEQ ID. NO. 25 and SEQ ID. NO. 26, SEQ ID. NO. 27 and SEQ ID. NO. 28, SEQ ID. NO. 29 and SEQ ID. NO. 30, and SEQ ID. NO. 31 and SEQ ID. NO. 32.
US10/769,579 1994-09-30 2004-01-30 Multiplex amplification of short tandem repeat loci Abandoned US20040137504A1 (en)

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