CA2013430A1 - Method for identifying human subjects by analysis of genetic material - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method for genetic typing of human DNA is disclosed. The method has utility in detecting microsatellite polymorphisms in DNA from a sample of human individuals selected from a population of individuals and in determining a human subject from a sample of biological material containing human DNA. The method examines polymorphisms of a (dT-dG)n VNTR region at the D11S35 locus on human chromosome 11. The method comprises the steps of isolating the human DNA, heat-denaturing the DNA
to form single strands, hybridizing synthetic DNA
oligomers as described herein to the denatured DNA so as to generate primer sites adjacent said (dT-dG)n regions on the DNA, amplifying the D11S35 VNTR region using the polymerase chain reaction method, and separating the amplified segments. Different individuals exhibit distinctive allelic patterns of separated segments, which can be used to confirm identities, pedigrees, etc., and in forensic studies. The sequences of preferred synthetic DNA oligomer primers, termed oligomers 780 and 781, have been determined and are disclosed herein.

Description

NETHOD F9R IDENTIFYING H~MAN BUBJECTS BY
ANALYBIS OF GENETIC MATERIAL ~ -Field of the Invention This invention pertains to the use of specific 5 oligonucleotides as probes for determining the source or -identity of human biological material. -Background of the Invention The human genome contains approximately 50~000 copies of an interspersed nucleotide repeat with the seq~ence (dT-dG:dA-dC)n, where n is an integer from about 10 to 60. Several of these repeats have variable lengths ~ ;
in different individuals, where allelic fragments of the repeats vary in size by multiples of 2 base pairs (bp) or more. Litt and Luty, Am. J. Hum. Genet. 44:397-401 -(1989); Weber and May, ~m. J. Hum. Genet. (Sup~l.) 43:A161 ~`
(1988). Generally, such repeats display extensive length ~
polymorphism in humans as variable numbers of tandem `
repeats (VNTRs).
Many highly polymorphic VNTRs found to date in human genomes tend to be distributed mainly in subtelomeric regions of human chromosomes. Unfortunately, genetic information located in such regions can be difficult to retrieve without performing haplotyping or ;
other difficult procedure and difficult to analyze accurately.
Summary of the Invention In an attempt to circumvent this problem, a search was made for highly polymorphic (TG) n ., microsatellite regions in interstltial regions of chromosomes.
A number of clones previously mapped to interstitial chromosomal regions were searched to ascertain if the clones also included highly polymorphic loci. A locus was found, designated DllS35, that contained at least one polymorphic (dT-dG) n microsatellite region. The DllS35 locus had been previously mapped to region llq22 on human chromosome 11. Maslen et al., Genomics 2:66-75 (1988). The DllS35 locus was found to -` 2~13~3`Q

have at least 6 alleles and a heterozygosity of nearly 90~. This finding suggested that highly polymorphic (TG) n microsatellites may be readily ascertained in interstitial genomic regions.
5Because the full information content of interstitial polymorphic loci such as DllS35 is more accessible for genetic analysis, these loci are especially useful, for example, in mapping genes for late-onset diseases and syndromes via affected sib-pair and affected relative-pair analyses.
To resolve the VNTRs at the DllS35 locus, a sample of human DNA i~ first denatured to form single- -ctranded DNA. After denaturation, the human DNA is ~ -~
annealed with two unique single-stranded oligonucleotide primers. The first primer is homologous with a portion of a unique DNA sequence on a first DNA strand flanking a first end of the (dT-dG)n microsatellite region at the DllS35 locus. The second primer is homologous with a portion of a unique DNA seguence on a second strand flanking a second end of the same (dT-dG)n microsatellite region. After annealing, the primers are extended using the polymerase chain reaction (PCR) technique using ~; ~
radiolabeled DNA precursers. Plural PCR cycles including : ;
heat denaturation of the DNA, annealing of the primers to ;
25 their complementary sequences on the human DNA, and ;~
extension of the annealed primers with DNA polymerase are ; ~;
employed to enormously increase the numbers of copies of the (dT-dG)n microsatellite repeat sequences adjacent the hybridized primers. The amplified copies are then electrophoresed on sequencing gels for sizing and interpretation of the resulting pattern. ~;
The preferred oligonucleotide primers, designated -~
oligomers 780 and 781, were custom-synthesized as described herein. Each primer had a known sequence 20 ;-; -nucleotides long, where each sequence corresponded to a portion of the sequence of a DllS35 locus present in the ~
genome of a recombinant phage. Although oligomers 780 and ;
781 are the preferred primers, other single-stranded DNA

20~3~30 oligomers having nucleotide sequences homologous to other portions of the unique sequences flanking the (dT-dG)n microsatellite repeat region at the DllS35 locus can al~o be used, where each primer has a length within the range of about 15 to about 30 nucleotides long.
Hence, one feature of the present invention comprises a method for detecting microsatellite ~ -~
polymorphisms in DNA from a sample of human individuals selected from a population of individuals. Steps in the method include isolating DNA from each individual in the sample; heat-denaturing the DNA; annealing single-stranded DNA oligomeric primers to the denatured DNA, wh~re the oligomeric primers are homologous to unique nucleotide sequences flanking the microsatellite (dT-dG) n repeats at the DllS35 locus; performing multiple cycles of the polymerase chain reaction on the primed DNA to amplify copies of microsatellite DNA sequences located adjacent the hybridized primers; and separating the amplified copies of DNA for visualization and interpretation. The amplified seguences comprise (dT-dG) n repeat sequences at the DllS35 locus on human chromosome 11. This method can be used, for example, for ascertaining a person's pedigree or genetic identity.
As another feature of the present invention, the method as described hereinabove can also be used for determining the identity of a human subject, wherein the human su~ject is suspected of being the source of a sample of biological material of unknown origin. Such a method has especial utility in forensic investigations.
As another feature of the present invention, the step of using the polymerase chain reaction instead of using Southern Blotting or related techniques renders the present much faster to perform than existing techniques of genetlc typing. Further, use of DNA sequencing gels to 1 35 resolve amplified sequences facilitates the speed of the i present method over existing methods.

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As another feature of the present invention, primers 780 and 781 have been sequenced as a preliminary step to custom-synthesis of each primer.
Brief Description of the Drawina ~-FIG. 1 shows the nucleotide sequence of a 196 bp region of the phage ~2-22 subclone including a ~T-G) 17 repeat used to design oligonucleotide primers used in the present invention.
FIG. 2 is an autoradiogram of a sequencing gel used to resolve PCR-amplified segments from genomic DNAs from members of a three-generation family with autosomal ;
dominant episodic ataxia, showing the allelic characteristic of the microsatellite (dT-dG) n repeat at the DllS35 locus, as determined via the present method.
Detailed Descrie~ion ~ ;~
The preferred flanking primers used herein were ;;
designated oligomers 780 and 781. Each was a synthetic DNA oligomer 20 nucleotides (nt) long. These oligomers were designed to have sequences complementary to portions of unigue genomic sequences flanking a (dT-dG) n microsatellite region at the DllS35 locus. The DllS35 locus is normally located on human chromosome 11.
After hybridization to a sample of human DNA
containing the DllS35 locus, oligomers 780 and 781 were used as primers for multiple cycles of the polymerase chain reaction (PCR). The PCR produced a selective amplification of the (dG-dC)n-containing sequences of at least a million fold. The PCR technique is described generally in Saiki et al., Science 239:487-491 (1988).
' 30 Oligomers 780 and 781 were synthesized as follows: ~-Using as a hybridization probe a commercially available alternating copolymer of poly-(dG-dT):poly-(dC-dA), a number of recombinant phage clones containing DNA
from the long arm of human chromosome 11 were screened.
See Frischauf et al., J. Mol. Biol. 170:827-842 (1983) for details on constructing the llq;16q chromosome-specific -~
library using the lambda replacement vector EMBL-3. The ~` ;

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screened recombinant phages used as described herein originated from such a library. See also Maslen et al., Genomics 2:66-75 (1988). ~ -The phage ~2-22, previously mapped to the DllS35 locus on the llq22 chromosomal region, Maslen et al., Genomics 2:66-75 (1988), gave a strong signal when hybridized with the poly (dG-dT):poly (dC-dA) probe. To perform these analyses, slot blots were performed wherein each slot contained 10 ng DNA from several selected clones of chromosome llq lambda phage. Maslen et al., Genomics ~:66-75 (1988). Each slot was probed with nick-translated poly (dG-dT):poly (dA-dC) (Pharmacia). Hybridization was performed overnight at 65C in 0.5 M sodium phosphate, pH
7.0, 7 % (w/v) SDS, 1 % (wlv) bovine serum albumin, where the solution lacked nonradioactive DNA. Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984).
Filters were washed at a maximum stringency of 0.1 X
SSC/O.l % SDS at 55C. Overnight autoradiography indicated that phage ~2-22 (DllS35) gave a strong signal.
A Sau 3A digest of phage ~2-22 was subcloned into the Sal 1 site of the plasmid vector pTZ18u. Colony filters containing subclones were probed with nick-translated poly (~G-dT):poly (dA-dC) as described above to identify subclones containing (TG) n microsatellites. One subclone found to contain (TG) n microsatellites was sequenced using a standard dideoxynucleotide sequencing protocol. Sanger et al., Proc. Natl. Acad. Sci. USA
74:5463-5467 (1977). The sequence of a 196 bp region of the ~2-22 subclone inc~uding a (T-G) 17 repeat (underlined) is shown in FIG. 1. Twenty-nucleotide segments flanking this repeat, shown boxed, were used to design oligomers 780 and 781. In choosing these specific segmen~s, the following criteria were considered in descending order of importance: (1) sequences containing four or more contiguous purines or pyrimidines or other simple sequences should be absent; (2) the GC contents of the two primers should match within +/- 2 nt; and (3) the primers should flank the (TG)n repeats as closely as possible.

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Although primers 780 and 781 were deemed to be the preferred primers derivable from the sequence shown in FIG. 1, primers having other unique sequences flanking the (dT-dG) n microsatellite repeat at the DllS35 locus are also possible. Generally, primers as used in ~he present invention should have a single-stranded length within the range of about 15 nucleotides to about 30 nucleotides, and preferably about 20 nucleotides long. Shorter primers tend to exhibit lesser specificity when hybridizing to human DNA; longer primers require higher hybridization temperatures and longer hybridization times. Also, longer primers are more expensive to custom-synthesize.
Using the sequence as shown in FIG. 1 as a guide, 20-nt primers 780 and 781 flanking the (TG)n microsatellite repeat region were synthesized on an Applied Biosystems Model 380A DNA Synthesizer and purified on a 12% denaturing acrylamide gel. The oligomer primer sequences were:
Oligomer 780: 5' ACA ATT GGA TTA CTA CTA GC 3' Oligomer 781: 5' TGT ATT TGT ATC GAT TAA CC 3' These primers were used to amplify the (dT-dG) n microsatellite region of locus DllS35 in samples of genomic DNA from family members and unrelated individuals.
Genomic DNA is prepared as described by Litt and White, Proc. Natl. Acad. Sci. USA 82:6206-6210 (1985).
After denaturation of the DNA to form single-stranded DNA, ~-the PCR method of Saiki et al. is used to amplify the (dT-dG) n microsatellite xepeat regions at DllS35 loci in the DNA. Amplifications were performed using 250-ng samples of genomic DNA. To perform the PCR, the thermostable Taq 1 DNA polymerase and additional reagents supplied in the GeneAmpT~ kit (Perkin Elmer-Cetus, Norwalk, CT) were used.
~ 35 Reaction mixtures were prepared according to the I instructions of the manufacturer, except that the total volume was decreased to 25 ~L and 3-5 ~Ci of ~-[32P]dCTP
was included in each reaction tube.

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~013~0 The PCR amplification method comprises multiple cycles of heat-denaturing the DNA, annealing the primers to their complementary sequences on the denatured DNA, and extension of the annealed primers using a DNA polymerase.
The preferred DNA polymerase is the thermostable DNA
polymerase purified from the thermophilic ~acterium Thermus aquaticus (~) which is stable to about 95C.
The required number of amplification cycles can vary, depending upon the amount of genomic DNA in the original sample and the amount of DNA required for subsequent analysis of the length distribution of the amplified portions thereof. In one test, thirt~v-seven cycles of amplification were performed manually wherein each cycle included a denaturation step for 1 min at 90-92C, an annealing step for 2 min at 40C, and a primer extension step for 2 min at 72C. The thirty-seven amplification cycles produced a sufficient amount of amplified sequences for visualization thereof on a DNA
sequencing gel.
To separate the amplified sequences for subsequent length-distribution analysis, aliquots of the DNA were electrophoresed on a DNA sequencing gel in alternate lanes. After electrophoresis, gels were autoradiographed for 1-3 days, usually without intensifying screens. Segment lengths were measured relative to size standards consisting of end-labeled Sau 3A fragments of pBR322 and/or DNA sequence ladders derived from a known sequence.
Under conditions as described hereinabove, oligomers 780 and 781 amplified polymorphic microsatellite (dT-dG) n sequences at DllS35 loci having at least six different alleles as seen in 17 unrelated individuals. As shown in Table 1, fifteen of these 17 individuals (88%) were heterozygous. Using the allele frequencies shown in Table 1, a polymorphism information content (PIC) value of 0.79 was calculated. Botstein et al., Amer. J. Hum.
- Genet. 32:314-321 (1980).

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Allele Size (nt) Number FIG. 2 shows an autoradiogram of a sequencing gel used to resolve (dT-dG)n microsatellite repeats amplified ~ ;
from yenomic DNAs from members of a three-generation family. The family has a history of autosomal dominant episodic ataxia. Each lane contains one or two relatively intense "major'l bands where each major band is closely associated with a cluster of up to eight less-intense minor bands spaced at one-nucleotide intervals.
Considering that each of the major bands represents an allele, codominant Mendelian inheritance is observed in the family shown in FIG. 2. Another study of three nuclear families with a total of 22 children confirmed that the polymorphism was inherited in a Mendelian fashion (data not shown).
Within a set of unrelated individuals, the value of n in the (dT-dG) n microsatellite repeat at the DllS35 locus was found to vary extensively. Indeed, as disclosed above, oligomers 780 and 781 revealed a highly polymorphic locus in human DNA with a heterozygosity of 88%. Hence, this pair of oligomers has utility in paternity testing and in other forensic procedures, such as identifying or ruling out individuals suspected of rape, tracing the source of a bloodstain, and identifying a badly disfigured corpseO In particular, any sample of biological material containing human DNA can be tested using the present method and oligomers 780 and 781 to help determine the origin of the sample. The present method is particularly useful with small samples because the PCR step amplifies the polymorphic sequences of interest to a concentration 2~3~3~
g sufficient for conventional analysis using gel electrophoresis.
The preferred method for detecting (TG)n microsatellite polymorphisms is via th~ use of primers 780 and 781 and the polymerase chain reaction (PCR) to amplify the repeat-containing region of interest located between the primer-annealing sites in genomic DNA. This has major advantages over the conventional Southern blotting technique used to detect restriction fragment length polvmorphisms (RFLPs) that are often used for forensic purposes. In particular, the new method as described herein is far more sensitive than Southern Blotting. In particular, 250-ng samples of human DNA have been used in the present method which is tenfold lower than required for Southern blotting. Since others have used the PCR
method to amplify extremely small quantities of human genomic DNA present in a single cell, it is very likely that, in the present method, the required sample size for analysis could be decreased hy at least several orders of magnitude, if necessary. Another advantage is that the technology described seems to be faster and more reliable than Southern Blotting.
As shown in Table 1, only six alleles have been found to date at the DllS35 locus. As a result, there is a small but calculable probability that two unrelated individuals having genomes probed using primers 780 and 781 will display the same genotype. This limitation can be minimized by using additional primer pairs which detect other highly polymorphic (TG) n microsatellites, such as primers 635 and 636. Litt and Luty, Am. J. Hum. Genet.
44:397-401 (1989). Since there is no overlap between segment sizes observed with these two primer pairs, it should be feasible to type individuals for both polymorphic loci by co-amplification of genomic DNAs with the products resolved on a single gel.
The results described hereinabove indicate that the DllS35 locus, previously mapped to llq2~l contains a microsatellite VNTR with at least six alleles and a PIC of ;

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20~30 about 0.78. These results also indicate that highly polymorphic (TG) n microsatellite VNTRs can be readily ascertained in genomic DNA regions where "classical"
VNTR's are scarce. Interestingly, another research group has described microsatellite VNTRs at interstitial genomic regions on other human chromosomes: APOA2 (PIC = 0.65;
lq21-q23) and APOC2 (PIC = 0.79; l9ql2-ql3.2). Weber and May, Amer. J. Hum. Genet. Sup~l. 43:A161 (1988). Because their full information content is inaccessible without haplotyping, VNTRs in interstitial chromosomal regions are especially useful in genetic mapping of "late onset"
disorders, such as Alzheimer's disease, via affected sib pair and affected relative pair analyses.
Having illustrated and described the principles of the present invention, it should be apparent to those of ordinary skill in the art that such embodiments may be modified in detail without departing from such principles.
I claim as my invention all such modifications as come within the true spirit and scope of the following claims.

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Claims (18)

1. A method for detecting microsatellite polymorphisms in DNA from a sample of human individuals selected from a population of individuals, the method comprising the steps:
(a) isolating DNA from each individual in the sample;
(b) heat denaturing the DNA to form single strands of DNA;
(c) annealing first and second DNA
oligonucleotide primers to the denatured DNA, where each first primer is homologous to a unique genomic sequence normally flanking a first end of a polymorphic (dT-dG)n microsatellite repeat sequence at a D11S35 locus in human DNA and each second primer is homologous to a unique genomic sequence normally flanking a second end of said polymorphic (dT-dG)n microsatellite repeat sequence;
(d) extending the annealed primers using a DNA
polymerase;
(e) repeating steps (c), (d), and (e) a multiplicity of cycles so as to amplify copies of microsatellite DNA adjacent the annealed primers; and (f) separating the amplified copies of microsatellite DNA to yield a pattern of separated copies of microsatellite DNA for each individual.

2. A method for detecting microsatellite polymorphisms as recited in claim 1 wherein the first and second DNA oligonucleotide primers have single-stranded lengths within the range of about 15 nucleotides to about 30 nucleotides.

3. A method for detecting microsatellite polymorphisms as recited in claim 1 wherein the first and second DNA oligonucleotide primers are oligomers 780 and 781, respectively.

4. A method for detecting microsatellite polymorphisms as recited in claim 1 as used for ascertaining the pedigree of an individual.

5. A method for detecting microsatellite polymorphisms as recited in claim 1 as used for determining the identity of an individual.

6. A method for determining a human subject from a sample of biological material containing human DNA, the method comprising the steps:
(a) isolating DNA from the sample and from the human subject, wherein the DNA from the sample and the human subject includes D11S35 loci each comprising at least one (dT-dG)n microsatellite repeat sequence;
(b) performing multiple cycles of a polymerase chain reaction comprising heat-denaturing the DNA from the sample and the subject, hybridizing first and second synthetic DNA oligomeric primers to the denatured DNA so as to generate primer sites flanking (dT-dG)n microsatellite repeat sequences at the D11S35 loci in the DNA from the sample and the subject, and extending the annealed primers using a DNA polymerase so as to amplify the number of copies of said (dT-dG)n microsatellite repeat sequences;
(c) separating the amplified copies of said (dT-dG)n repeat sequences so as to reveal an allelic pattern of said sequences for the sample and an allelic pattern of said sequences for the subject; and (d) comparing the allelic pattern obtained from the sample to the allelic pattern obtained from the subject.

7. A method for determining a human subject as recited in claim 6 wherein the first synthetic DNA
oligonucleotide primer is homologous to a unique genomic sequence normally flanking a first end of a polymorphic (dT-dG)n microsatellite repeat sequence at a D11S35 locus in human DNA and the second synthetic DNA oligonucleotide primer is homologous to a unique genomic sequence normally flanking a second end of said polymorphic (dT-dG)n microsatellite repeat sequence.

8. A method for determining a human subject as recited in claim 7 wherein the first and second synthetic DNA oligonucleotide primers each have single-stranded lengths within the range of about 15 nucleotides to about 30 nucleotides.

9. A method for determining a human subject as recited in claim 8 wherein the first and second synthetic DNA oligonucleotide primers each have single-stranded lengths of about 20 nucleotides.

10. A method for determining a human subject as recited in claim 6 wherein the first and second synthetic DNA oligonucleotide primers are oligomers 780 and 781, respectively.

11. A method for determining a human subject as recited in claim 6 wherein said amplified copies are separated by electrophoresis on a DNA sequencing gel.

12. A method for determining a human subject as recited in claim 6 wherein the annealed primers are extended in the presence of radiolabeled DNA precursers so as to radiolabel the amplified copies of said (dT-dG)n repeat sequences.

13. First and second oligonucleotide DNA primers having the properties of primers 780 and 781.

14. First and second oligonucleotide DNA primers as recited in claim 13 for initiating polymerase chain reaction amplification of (dT-dG) n repeat sequences present in a sample of human DNA.

15. First and second oligonucleotide DNA primers as recited in claim 13 wherein the first primer has a nucleotide sequence 5' ACA ATT GGA TTA CTA CTA GC 3' and the second oligonucleotide primer has a nucleotide sequence 5' TGT ATT TGT ATC GAT TAA CC 3'.

16. First and second oligonucleotide DNA primers as recited in claim 13 that hybridize to nucleotide sequences in human DNA flanking (dT-dG)n repeat sequences at D11S35 loci.

17. An oligonucleotide DNA primer having the nucleotide sequence 5' ACA ATT GGA TTA CTA CTA GC 3'.

18. An oligonucleotide DNA primer having the nucleotide sequence 5' TGT ATT TGT ATC GAT TAA CC 3'.