WO1991010748A1 - Sequences d'adn provenant de loci genomiques humains specifiques utiles a l'identification d'individus - Google Patents

Sequences d'adn provenant de loci genomiques humains specifiques utiles a l'identification d'individus Download PDF

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WO1991010748A1
WO1991010748A1 PCT/US1991/000196 US9100196W WO9110748A1 WO 1991010748 A1 WO1991010748 A1 WO 1991010748A1 US 9100196 W US9100196 W US 9100196W WO 9110748 A1 WO9110748 A1 WO 9110748A1
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dna
vntr
oligonucleotide
primers
region
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PCT/US1991/000196
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Tim P. Keith
Jen-I Mao
Stanley D. Rose
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Collaborative Research, Inc.
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates in general to methods and products used for identity testing and, in particular, to oligonucleotide probes and primers and methods of using such probes and primers.
  • the methods and products of this invention are particularly useful for paternity or forensic applications.
  • Regions of the human genome that are quite variable in different individuals have been described (Wyman & White, 1980, Proc. Nat' 1 ⁇ Acad. Sci. USA 77: 6754- 6758; Donis-Keller et al, 1987, Cell 51: 319-337). Many of these regions of variability or "polymorphisms" are observed by cleaving human DNA with one of a variety of sequence-specific restriction endonucleases, separating the cleaved fragments by size on a gel, transferring the DNA to a membrane, and hybridizing the DNA with probe which has been labeled with a reporter group such as 32P.
  • a reporter group such as 32P.
  • RFLP restriction fragment length polymorphism
  • VNTR very short tandem repetitive DNA sequences
  • VNTR locus One of the earliest descriptions of such a VNTR locus was published by Jarman et al. (1986, EMBO Jour. 5: 1857-1863) and described the 3' flanking region of the human beta-globin locus which contains in different individuals between 70 and 450 copies of a 17 bp repeat. Other loci containing such VNTRs have been described since that time. At the present time, over .30 such VNTR loci have been described in the literature.
  • This invention provides nucleic acid or oligonucleotide probes and primers capable of hybridizing selectively to VNTR loci and useful for determining identity.
  • the variability of a VNTR region from one individual to another can be so high the pattern of numbers of repeats at several different genetic loci revealed by probing with a panel of different VNTR probes can be unique for a particular individual. If the panel of probes is large enough, the identity of a sample can be determined with confidence.
  • these patterns can be used in forensic applications, in establishing paternity, and in any application in which ability to unambiguously identify an individual is important.
  • This invention involves the discovery that a particular. group of loci, D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129, have at least one region containing a variable number of tandem repeats (hereinafter VNTR region) and the subsequent DNA sequencing of these VNTR regions and regions which flank the VNTR regions.
  • VNTR region a variable number of tandem repeats
  • the identification of these VNTR loci along with the sequence information provides sufficient basis for preparing oligonucleotide probes and extension primers.
  • These probes and primers are specially designed for use in assays involving the comparative study of two samples of DNA and the determination of identity or nonidentity between the source of the two samples.
  • the oligonucleotide probes of the invention are capable of selectively hybridizing to a VNTR region of a locus selected from the group consisting of D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129. These oligonucleotide probes may be of a size and specificity so as to hybridize only to a single repeat region within the VNTR, to two or more contiguous repeat regions within the VNTR or to the entire VNTR region. The probes of the invention also may be of a size and specificity so as to hybridize only to regions which flank the VNTR regions or to both a flanking region and a VNTR region contiguous with one another.
  • the invention also provides primers for initiating the synthesis of an extension product along a strand of DNA.
  • These primers are oligonucleotides capable of hybridizing to at least a portion of the flanking region of a VNTR region of a locus selected from the group consisting of D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129, and further are capable of initiating the synthesis of an extension product including at least a portion of the VNTR region in the presence of the appropriate nucleoside triphosphates and a suitable polymerization agent.
  • These primers are not capable of hybridizing to only the VNTR region under Polymerase Chain Reaction (hereinafter PCR) extension conditions.
  • the oligonucleotide primers preferably are provided in pairs adapted to interact with a common segment of DNA to amplify a VNTR region of a locus.
  • a first oligonucleotide primer of the pair has a sequence complementary with at least a portion of a VNTR flanking region at one end of a VNTR region of a first strand of a locus.
  • the second oligonucleotide primer of the pair has a sequence complementary with at least a portion of the VNTR flanking region of a second strand of the locus complementary to the first strand at the opposite end of the VNTR region.
  • the pair of primers are non-homologous to and non-complementary with one another.
  • the primers are not capable of hybridizing to only VNTR regions under PCR extension conditions.
  • a method for determining the genetic identity between two samples of DNA is provided.
  • a first sample of DNA is treated to produce fragments of DNA, the fragments having at least one VNTR region of the locus selected from the group consisting of D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129 and the at least one VNTR region being present in its entirety as a contiguous piece of DNA.
  • the DNA fragments, or an amplified product thereof are separated based at least in part upon the size of the VNTR regions thereby producing a fingerprint of the first sample.
  • the foregoing procedure is repeated with a second sample of DNA, and the fingerprint of the first sample and second sample of DNA are compared to establish or exclude identity of the DNA samples.
  • a Southern Blot Assay and the probes of the invention are used to create the fingerprint.
  • a first sample of DNA is digested with enzymes to produce fragments of DNA.
  • the digested fragments are separated in a gel under conditions which allow the fragments of DNA to move to positions within the gel based upon their size, thereby creating a pattern of bands.
  • the DNA within the gel then maybe transferred to a substrate maintaining the pattern of bands.
  • the DNA on the substrate then can be contacted with the labelled oligonucleotide probes of the invention to create the fingerprint.
  • the DNA can be dried within the gel and contacted directly with an oligonucleotide probe(s). The steps are repeated with a second sample of DNA, and the fingerprints created are compared.
  • PCR is employed using primers of the invention to create the fingerprints.
  • at least one VNTR region of a first sample of DNA is amplified, the VNTR region being within a locus selected from the group consisting of D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129.
  • the amplified DNA then is separated from other DNA in the sample based at least in part upon the size of the amplified DNA, and a fingerprint is created.
  • the DNA is separated in a gel based upon size and the fingerprint is created by staining the DNA in the gel.
  • the steps are carried out with a second sample of DNA to create a second fingerprint, and the two fingerprints are compared to establish or exclude iden ity.
  • the foregoing methods of the invention are carried out in connection with at least two different VNTR loci, and most preferably three or more.
  • FIG. 1 is a photograph showing the gel electrophoresis separation of five DNA samples, portions of which were amplified using primers capable of hybridizing with regions which flank the VNTR region of locus D20S15.
  • This invention relates to probes and primers capable of hybridizing with various regions within or flanking a particular group of VNTR loci and methods of using these probes or primers for determining identity.
  • oligonucleotide refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, synthetic or natural, and preferably more than three. The exact size of the molecule may vary according to its particular application.
  • probe refers to an oligonucleotide capable of hybridizing to a strand of DNA in connection with determining the existence and location of the strand of DNA.
  • the probes of the invention typically are labelled, isotopically or non-isotopically.
  • labelled nucleoside triphosphates may be used in constructing the probes or the probes may be labelled post-construction.
  • the label may be, for example, a radioactive label, an enzyme, or a fluorescent chromophore.
  • primer refers to an oligonucleotide which, when hybridized to a strand of DNA including a complementary or substantially complementary sequence, is capable of initiating the synthesis of an extension product in the presence of the appropriate nucleoside triphosphates and a suitable polymerization agent.
  • the primer is an oligoribonucleotide and most preferably is a oligodeoxyribonucleotide.
  • the primer may be other than a ribonucleotide or deoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of an extension product.
  • the exact length of the primer will depend upon many factors, including the degree of specificity of hybridization required, temperature of the annealing and extension reactions and the source and structure of the primer.
  • the term "capable of hybridizing to” refers to all methods of sequence specific pairing between the probe or primer and a strand of DNA. It includes situations in which there is complete complementation between the molecules hybridizing to one another, and situations when there is less than complete complementation.
  • primers and DNA sequences shall mean a primer homologous or substantially homologous to a DNA sequence or a primer complementary or substantially complementary to a DNA sequence.
  • complementary when referring to two oligonucleotides or an oligonucleotide and a DNA sequence shall mean sequences which are completely complementary as well as those sequences which are substantially complementary (i.e. less than complete complementation — some mismatches of base pairs).
  • substantially complementary it is meant that the oligonucleotides are capable of hybridizing to one another to perform their intended functions in the invention under appropriate conditions.
  • oligonucleotides or an oligonucleotide and a DNA sequence shall mean sequences which are identical or which are substantially identical. By substantially identical, it is meant that the oligonucleotide may have some mismatches but is still is capable of performing its intended function in the invention under the appropriate conditions.
  • fingerprint refers to a pattern derived from separated DNA fragments prepared from a source of DNA. An example of such a fingerprint is a pattern produced by separating fragmented DNA based upon size within a gel using gel electrophoresis.
  • oligonucleotide primers and probes of the invention may be prepared using any method, such as, for example, methods using phosphotriesters and phosphodiesters well known to those skilled in the art.
  • diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage and Caruthers 1981, Tetrahedron Lett. 22: 1859-1862.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U. S. Patents 4,458,066 and 4,500,707. It also is possible to use primers or probes which have been isolated from a biological source (such as restriction endonuclease digest of plasmid DNA) .
  • the samples of DNA used in the methods of this invention can be from any source of nucleic acid, in purified or non-purified form.
  • the DNA can be extracted, for example, from blood or tissue material using a variety of conventional techniques such as those described by Maniatis et al. Molecular Cloning: A Laboratory Manual, (New'York: "Spring Harbor Laboratory, 1982) pp. 280-281.
  • the degree of certainty with which two DNA samples can be said to be from the same individual depends on the heterozygosity of the particular polymorphisms under investigation and on the distribution of particular alleles within different populations (E.S. Lander, 1989, Nature 339: 501-505). For example, some alleles may be very common in certain isolated populations. In that case, the presence of the same allele in two different DNA samples would not indicate a high probability that those two DNA samples were from the same individual (the presence of different alleles in the two samples, however, would indicate a high probability of exclusion of identity).
  • a database of information on allele frequencies can be obtained. In general, a database with information from at least 1000 chromosomes for each loci for each ethnic group would be ideal for DNA typing studies.
  • VNTR polymorphisms in general, exhibit very high heterozygosities because very many different alleles occur. A difference of one repeat unit constitutes a different allele. (Balazs et al, 1989, supra) .
  • the DNA sequences of this invention are useful for the application of rapid, sensitive methods for the determination of identity or exclusion of identity between the genetic material in two or more
  • DNA sequences permit both the synthesis of labeled oligonucleotide probes to identify VNTR alleles in Southern Blot fingerprints and, alternatively, the synthesis of PCR primers so that VNTR alleles can be analyzed without the need for labeled probes and Southern blot fingerprints.
  • Analysis of the genetic loci described by the DNA sequences herein can establish identity or exclude identity between DNA samples with a high degree of certainty. For example, because the loci describe here exhibit very high heterogosities a combination of the data resulting from the use of all of the probes described here on actual DNA samples should provide an average probability of exclusion of identity in excess of 99.95% and an average probability of identity of better than 9.4 x
  • Nucleic acid probes capable of hybridizing to over 180 different loci were described by Donis-Keller et al., cited supra, the contents of which are incorporated herein by reference.
  • the DNA probes CRI-L159, CRI-L336, CRI-L355, CRI-L427, CRI-L1077 and CRI-R365 were selected from this large group of probes as potentially including VNTR regions. These probes were prepared and isolated using the techniques described by Donis-Keller et al. Subsequent to selection, the probes were sequenced using conventional techniques.
  • the DNA sequences of the cloned loci were determined by the dideoxy chain-terminating method (Chen & Seeburg, 1985, DNA 4: 165-170; Tabor & Richardson, 1987, Proc. Nat. Acad. Sci. USA 84: 4767-4771). From sequence analysis of these probes, the DNA sequences of seven repeated VNTR regions and the surrounding unique regions were identified.
  • the Southern blot is a reliable approach to measuring VNTR lengths and thereby identify alleles.
  • a first DNA sample is digested with enzymes to produce fragments of DNA.
  • enzymes are capable of cutting the sample of DNA into fragments containing VNTR regions in their entirety as contiguous pieces of DNA. Examples of such enzymes include Alul, Pstl, and Haelll. The selection of enzymes depends upon the loci being examined. Table 1 shows the preferred enzymes for each locus and/or type of probe. Table 1
  • the digested fragments are separated in a gel under conditions which allow the DNA fragments to be separated based upon size.
  • the gel is selected based upon such factors as the size of DNA being separated and ability to withstand conditions necessary for separation. Examples of gels include agarose and polyacrylamide gels.
  • the conditions, which allow the DNA fragments to be separated, can be determined by one of ordinary skill in the art.
  • the DNA within the gel then is transferred to a substrate using conventional techniques, e.g. blotting or the gel can be dried andthe DNA can be contacted directly with a probe while it is within the dried gel (Ali and Wallace Nucleic Acid Research Vol. 16, No. 17 (1988), pp. 8487-8496.
  • the substrate is made of a substance which is capable of maintaining the pattern of bands of DNA transferred from the gel while not interfering in the hybridization process. Examples of such substrates include nitrocellulose filters and nylon membranes.
  • the DNA on the substrate is then contacted with a labelled oligonucleotide probe to create a fingerprint of the sample.
  • the DNA sequences provided in this invention permit the use of synthetic DNA segments as probes.
  • Such synthetic DNA is designed to be long enough to provide specificity of hybridization to the desired region, but short enough to be synthesized economically.
  • the probe can be labelled by a variety of means. For example, it can be labeled at its 5' end by T4 polynucleotide kinase (Maniatis, Fritsch, & Sambrook, "Molecular Cloning — a Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 122-127), or throughout most of its length by amplification according to the PCR method in the presence of labeled dNTPs as described in U. S. patents 4,683,195 and 4,683,202. The contents of both patents are hereby incorporated by reference.
  • the probe is labeled by a nonisotopic method, thereby providing long shelf-life and stability.
  • an oligonucleotide could be labeled with an enzyme such as alkaline phosphatase (Jablonski et al, 1986, Nucl. Acids Res. 14: 6115-6128).
  • alkaline phosphatase Jablonski et al, 1986, Nucl. Acids Res. 14: 6115-6128.
  • After hybridization to the DNA sample its presence would be detected by action of the enzyme on a substrate to produce a signal.
  • substrates provide visual signal by absorption of certain frequencies of visible light (Edman et al, 1988, Nucl. Acids Res. 16: 6235), or they expose film by emission of light
  • oligonucleotide probes also contain no contaminating labelled vector or bacterial DNA sequences. Thus, they can be used to analyze DNA samples which are contaminated by bacteria.
  • oligonucleotide probe Several parameters can be adjusted to insure that a single locus is detected by a particular oligonucleotide probe.
  • the length and specificity of the probe may be varied, the probe may consist of a mixture of probes which vary in sequence at one or a few nucleotide positions, and the stringency of hybridization may be varied, for example, by altering temperature and formamide concentrations as is known in the art.
  • the oligonucleotide probes of the invention are homologous or complementary to only a portion (not all) of any of the sequences of Examples 1-7. This portion may correspond to only a VNTR region, only a flanking region or both a flanking region and a VNTR region contiguous with one another.
  • the probes may also be of a size corresponding to a single repetitive sequence in a VNTR region. This type of probe may hybridize in multiple locations in the VNTR region providing increased sensitivity because of the increased number of labelled sites.
  • the probe in one preferred embodiment may correspond to a portion of or to the whole of any single tandem repeat of a VNTR region.
  • the probe may correspond to a contiguous region of two tandem repeats, i.e., the probe would hybridize to and overlap two adjacent single repeats.
  • the probe in another preferred embodiment is of a size corresponding to more than one repetitive sequence, but preferably of a size corresponding to less than five repetitive sequences,
  • the oligonucleotide probes are of a size large enough to provide the required specifity but are generally of a length which can be synthesized economically.
  • the preferred probes are 15-60bp in length, most preferred 18-40bp in length.
  • the probes should be sufficiently non-homologous to each other so that one probe will not hybridize to two different loci.
  • One of ordinary skill in the art will know how to construct probes based on the information provided herein.
  • a pair of primers may be used to amplify the VNTR region of a single VNTR locus or two or more pairs may be used to amplify (simultaneously or otherwise) the VNTR regions of two or more loci.
  • PCR primers hybridize to unique single-copy DNA sequences flanking the VNTR repeated regions. Since recent studies have shown that some VNTR repeats are themselves found within other moderately repetitive DNA regions (Armour et al. , 1989, Nucl. Acid. Res. 17: 4925-4935), it may be necessary in some cases to test PCR primers homologous to several different flanking DNA sequences to insure that a primer is selected from a unique region and that a single PCR amplified product is produced.
  • the unique single copy (flanking region) amplified along with the VNTR region should be as small as possible, or at least of a size that will not interfere with the detection of the number of repeat sequences in the VNTR regions of compared DNA samples.
  • the unique sequence amplified is 1000 base pairs, detecting the difference in the VNTR regions of two samples of DNA, one with a VNTR size of 200 and another with a
  • VNTR size of 240 would be difficult.
  • the unique single copy DNA will comprise less than 50 percent of the entire portion amplified.
  • the unique sequence amplified is less than 100 base pairs and most preferably between 30 and 60 base pairs. This provides sufficient priming template at either end of the VNTR regions (primers typically being of a size between 15 and 30 nucleotides) , yet is small enough so as to not interfere with the ability to distinguish VNTR regions which are close in size.
  • the limit on the size of the .unique sequence amplified will depend upon the size of the repetitive sequence tested and upon the frequency distribution of the number of repeats in the VNTR locus of the populations tested. The probes of the invention may be used to determine this frequency distribution in the population.
  • the probes can be used to isolate the VNTR locus of various individuals within the population. These isolated VNTR regions then can be seguenced to determine the number of the various repeats. This information can be compiled in a database to form a profile representative of the frequency distribution of the repeats in the population. The upper limit on the size of the unique sequence amplified then could be determined by one of ordinary skill in the art.
  • the pairs of oligonucleotide primers preferably are adapted to interact with a common segment of DNA containing a VNTR region within a locus selected from the group consisting of D18S17, D1S47, D20S15, D21S112, D6S22, and D11S129.
  • One of the pair has a sequence complementary with at least a portion of the flanking region at one end of the VNTR region of a first DNA strand of the selected locus.
  • the other of the pair has a sequence complementary to at least a portion of the flanking region of a second DNA strand (complementary to the first strand) at the opposite end of the VNTR region of the selected locus.
  • the pair of primers should be nonhomologous and noncomplementary with each other.
  • the primers preferably do not hybridize to only the VNTR regions under extension conditions. While the primers may be complementary to a contiguous flanking region and VNTR region, there preferably is insufficient complementation with the VNTR region to permit extension priming at other than the ends of the VNTR regions (i.e. no priming within a VNTR region under PCR extension conditions). Thus, the primers are preferably nonhomologous and substantially noncomplementary to the nucleotide sequences of the VNTR regions of Examples 1-7, below. Most preferably the primers are complementary or homologous to at least a portion of the flanking sequences of Examples 1-7.
  • EXAMPLE 1 The following DNA sequence includes flanking regions around the locus D18S17 detected by probe CRI-L159-1 on human chromosome 18 and copies (denoted 1-7; additional unsequenced copies are present betwee seguenced copies 3 and 4 — this is indicated by a dashed line) of th repeated region. Repeated units are aligned to maximize sequence hom
  • EXAMPLE 2 The following DNA sequence includes flanking regions around the V locus D18S17 detected by probe CRI-L159-2 on human chromosome 18 and s copies (denoted 1-7; ad itional unsequenced copies are present between seguenced copies 4 and 5 — this is indicated by a dashed line) of the repeated region. Repeated units are aligned to maximize sequence homo
  • CTTCCCTCAC CCAAGGGCCT 6 CTTCCCTCAC CCAAGGGCCT.
  • CCTGCCT 6 CTTCCCTCAC CCAAGGGCCT.
  • EXAMPLE 3 The following DNA sequence includes flanking regions around th locus D1S47 detected by probe CRI-L336 on human chromosome 1 and twel copies (denoted 1-12; additional unsequenced copies are present betwe sequenced copies 5 and 6 — this is indicated by a dashed line) of th repeated region. Repeated units are aligned to maximize sequence hom
  • EXAMPLE 4 The following DNA sequence includes flanking regions around the V locus D20S15 detected by probe CRI-L355 on human chromosome 20 and thi copies (denoted 1-13; additional unsequenced copies are present betwee seguenced copies 8 and 9 — this is indicated by a dashed line) of the repeated region. Repeated units are aligned to maximize sequence homo
  • EXAMPLE 5 The following DNA sequence includes flanking regions around the locus D21S112 detected by probe CRI-L427 on human chromosome 21 and fourteen copies (denoted 1-14; additional unsequenced copies are pres between seguenced copies 6 and 7 — this is indicated by a dashed lin the repeated region. Repeated units are aligned to maximize sequence homology.
  • EXAMPLE 6 The following DNA sequence includes flanking regions around the VN locus D6S22 detected by probe CRI-L1077 on human chromosome 6 and ten copies of the repeated region (denoted 1-10; this locus in the individu from which this clone was derived carried only these ten repeat copies) Repeated units are aligned to maximize sequence homology.
  • EXAMPLE 7 The following DNA sequence includes flanking regions around the V locus D11S129 detected by probe CRI-R365 on human chromosome 11 and el copies of the repeated region (denoted 1-11; this locus in the individ from which this clone was derived carried only these eleven repeat cop Repeated units are aligned to maximize sequence homology.
  • the sizes of the VNTR alleles at the human chromosome 20 locus D20S15 which are homologous to the human DNA in probe CRI-L355 are determined in five randomly selected individuals by the PCR technique.
  • DNA was prepared from peripheral blood cells in blood taken from five randomly selected individuals according to standard methods known in the art (see Bell et al., 1981, Proc. Nat'l. Acad. Sci. USA 78: 5759-5763; or Newton et al., 1988, Nucl. Acid. Res. 16: 8233-8243).
  • Synthetic oligonucleotide primers of the following sequence were designed to amplify the VNTR region of the locus homologous to probe CRI-L355 by PCR, and they were synthesized by Operon Technologies (San Pablo, CA) for applicants:
  • Primer #1 5' ACAATCTATACTACTAGA 3'
  • ejach sample was applied to a 2% agarose gel in Tris-acetate-EDTA buffer (Maniatis, Fritsch, & Sambrook, "Molecular Cloning — a Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, N.Y. , pp. 150-162) and subjected to electrophoresis at about 100 volts for about four hours.
  • the DNA was visualized by staining the gel with ethidium bromide (0.5 ug/ml) and subjecting it to illumination with uv light according to standard methods known in the art. As shown in Fig. 1, the DNA from each of the five individuals contains one or two amplified products, and these amplified products differ in migration position in the gel.
  • each individual carries a different number of sequence repeats at the D20S15 locus, and each individual carries two such loci, one on each of his chromosome 20 copies.
  • Both alleles in a single individual are not always visible, perhaps because they migrate at the same or nearly the same position in the gel or because one allele contains so many repeated units as to yield an inordinately large and poorly amplified product. Nevertheless, many of the individuals can be distinguished one from another due to the pattern of their alleles at the D20S15 locus. Clearly, caution must be exercised in interpreting results in which both alleles are not visible. Conclusions cannot be based on the apparent absence of that band.
  • different electrophoresis conditions including changes in the % agarose, use of a polyacrylamide gel, length of time of electrophoresis, or the type of buffer, may be used to reveal the second band in many cases.

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Abstract

Nouveau procédé permettant de déterminer l'identité génétique entre deux échantillons. Le procédé permet de classer par ordre de grandeur les régions VNTR des loci précités pour des échantillons d'ADN, lesquels peuvent être utilisés afin d'établir ou d'exclure une identité entre des échantillons. La détermination utilise avantageusement des sondes ou des amorces d'oligonucléotides capables de s'hybrider avec diverses régions de loci sélectionnées dans le groupe composé de D18S17, D1S47, D20S15, D21S112, D6S22, et D11S129.
PCT/US1991/000196 1990-01-16 1991-01-09 Sequences d'adn provenant de loci genomiques humains specifiques utiles a l'identification d'individus WO1991010748A1 (fr)

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Cited By (2)

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WO1992013968A1 (fr) * 1991-02-07 1992-08-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sondes hypervaribles a specificite monolocus
FR2680520A1 (fr) * 1991-08-22 1993-02-26 France Etat Armement Procede de detection de nouvelles regions hypervariables dans une sequence d'adn, sequences de nucleotides constituant des sondes d'hybridation et leur application biologique.

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US4965188A (en) * 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4987066A (en) * 1986-11-07 1991-01-22 Max Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Process for the detection of restriction fragment length polymorphisms in eukaryotic genomes
US4994368A (en) * 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays

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US4965188A (en) * 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4987066A (en) * 1986-11-07 1991-01-22 Max Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Process for the detection of restriction fragment length polymorphisms in eukaryotic genomes
US4994368A (en) * 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays

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AMERICAN JOURNAL OF HUMAN GENETICS, Vol. 32, issued 1980, BOTSTEIN et al., "Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms", pages 314-331. *
EMBO JOURNAL, Volume 5, No. 8, issued 1986, JARMAN et al, "Molecular Characterization of a Hypervariable Region Downstream of the Human-Globin Gene Cluster", pages 1857-1863. *
NUCLEIC ACIDS RESEARCH, Volume 16, No. 17, issued 1988, ALI et al, "Intrisic Polymorphism of Variable Number Tandem Repeat Loci in the Human Genome", see pages 8487-8496. *
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PROC. NATL. ACAD. SCI., Volume 79, issued June 1982, (Washinton, D.C.), EPPLEN et, "Base Sequence of a Cloned Snake W-Chromosome DNA Fragment and Identification of a Males Specific Putative mRNA in the Mouse", pages 3798-3802. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013968A1 (fr) * 1991-02-07 1992-08-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sondes hypervaribles a specificite monolocus
FR2680520A1 (fr) * 1991-08-22 1993-02-26 France Etat Armement Procede de detection de nouvelles regions hypervariables dans une sequence d'adn, sequences de nucleotides constituant des sondes d'hybridation et leur application biologique.
US5556955A (en) * 1991-08-22 1996-09-17 Etat Francais Represente Par Le Delegue General Pour A'armement Process for detection of new polymorphic loci in a DNA sequence, nucleotide sequences forming hybridization probes and their applications

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