WO1992013968A1 - Sondes hypervaribles a specificite monolocus - Google Patents

Sondes hypervaribles a specificite monolocus Download PDF

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Publication number
WO1992013968A1
WO1992013968A1 PCT/EP1992/000269 EP9200269W WO9213968A1 WO 1992013968 A1 WO1992013968 A1 WO 1992013968A1 EP 9200269 W EP9200269 W EP 9200269W WO 9213968 A1 WO9213968 A1 WO 9213968A1
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hypervariable
dna sequence
dna
eukaryotic
probes
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PCT/EP1992/000269
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English (en)
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Jörg EPPLEN
Hans Zischler
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO1992013968A1 publication Critical patent/WO1992013968A1/fr

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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
    • C12Q1/6876—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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 relates to monolocus-specific hyper ⁇ variable probes which can be used as probes in hybridization assays for the detection of specific hypervariable single copy loci (monoloci) in eukaryotic chromosomal DNA.
  • the haploid human genome comprises about 3.5 x 10 9 informa ⁇ tion units. Each unit can occur in four different conforma ⁇ tions (as four different nucleotides) : adenine (A) , cytosine (C) , guanosine (G) or thymidine (T) . Thus, theoretically there are about 4 3 5 00000000 (corresponding to about 10 2000000000 - possibilities for a genome of this size. This dimension can be understood better by comparing it to the volume of the universe which is about 10 108 A 3 (cubic Angstrom) . More then 90 percent of the diploid cell nucleus with about 7 x 10 9 nucleotides is a "genetic desert", i.e.
  • genes appear as "small oases in the barren stretch of desert".
  • genes are in the center of the interest of the present attempts for a total structural analysis of the human genome. However, they are usually not colinear with the DNA double strand but composed of "exons" (coding regions) and “introns” (noncoding regions) .
  • the en ⁇ visaged total sequencing of the human genome will provide a lot of information, the meaning of which cannot be reliably foreseen.
  • the established data bases already contain a large amount of intron sequences. These predominantly contain se ⁇ quences which only occur once per haploid genome, i.e. mono ⁇ locus elements.
  • the probes are either chemically synthesized oligonucleotides labelled at their 5'-end with 32 P-dATP or labelled with a nonradioactive reporting molecule such as digoxigenine.
  • the repetitive character of the oligonucleotide probes guarantees that they simultaneously hybridize to many parts of the genome. Thus, such probes inevitably provide a very complex pattern of bands which is also designated as a multilocus profile; see (1) to (4) .
  • An even distribution has been proven for the most informative probe in man, i.e. for (CAC) 5 /(GTG) 5 ; see (12).
  • the allelic bands of a particular locus in diploid cell nuclei appear as single bands in the resolvable part of the DNA fingerprint band pattern or as two bands.
  • Multilocus DNA fingerprints are used for determination of identity or kin ⁇ ship, e.g. in forensic science (5) , paternity determination (6) or animal and plant breeding (7) , (8) .
  • complex multilocus DNA fingerprints are not always particularly suitable for the analysis of individual genomic loci, e.g. in certain genetic diseases or various diseases based on so ⁇ matic changes such as translocation in certain eukaryotic chromosomes.
  • locus-specific DNA se ⁇ quences such as alleles of a particular gene, do usually not show heterogeneity that is sufficient for the above-men ⁇ tioned purposes (it can be considered as having only low in- formativity) .
  • the technical problem underlying the present invention is to provide DNA probes which can be reliably used in the determination of identity of given individuals or kinship and in the chromosomal analysis of specific loci in eukaryo ⁇ tic diploid cells, but which disclose less complex banding patterns in a genetic fingerprint (or blot) . These patterns can then be more easily and reliably interpreted. Further ⁇ more, they allow the identification of particular loci and their correlation with e.g. particular diseases.
  • hypervariable probes also designated as "monolocus probes" being capable of hybridizing with a chromosomal eukaryotic DNA sequence, said eukaryotic DNA sequence having the following features: (a) it only occurs as a single copy per haploid genome (mo ⁇ nolocus) ;
  • the probes of the present invention are locus-spe ⁇ cific.
  • locus specific probe refers to synthetic or cloned hypervariable probes, such as the chromosomal eukaryotic DNA sequences as characterized in the present invention, frag ⁇ ments thereof and oligonucleotides derived therefrom, said probes having a sufficient selectivity and specificity to hybridize with a given DNA sequence of a target organism.
  • the length of the oligonucleotides of the present invention i.e. of the pro ⁇ bes of the present invention, that is required for a spe ⁇ cific hybridization with the DNA of a given organism prim ⁇ arily depends on the complexity of the genome of this organ ⁇ ism.
  • the haploid genome comprises about 3.5 x 10 9 nucleotides, so that for the investigation of human target DNAs the oligomeric nucleotides of the pre ⁇ sent invention preferably have a length of at least about 18 nucleotides, preferably of at least about 20 nucleotides.
  • the cloned probes are hybridized under stringent conditions appropriate to yield locus-specific signals.
  • hypervariable probe refers to the chromosomal eukaryotic DNA sequences as characterized in the present in ⁇ vention, fragments thereof and oligonucleotides derived the ⁇ refrom.
  • closely adjacent as used throughout this specifi ⁇ cation means that the probe is located on the same restric ⁇ tion fragment as the hypervariable DNA sequences after di-area with enzymes recognizing 4 bases, such as Hinfl, Haelll, Alul or Mbol.
  • hypovariable DNA sequence refers to DNA se ⁇ quences that exceed the normal variability (polymorphism) by several orders of magnitude.
  • the term "70% heterozygosity rate" means that 70% or more of the individuals investigated for this par ⁇ ticular sequence show two discernable fragment lengths per locus.
  • the heterozygosity rate is at least 90%.
  • a heterozygosity rate of at least 90% provides surprisingly higher inter- individual resolution capabilities of the probes of the present invention.
  • the advantage of the monolocus probes of the present inven ⁇ tion over the multilocus probes of the prior art is that the alleles of a specific locus can be investigated separately because two allelic signals (corresponding to allelic forms) appear in a gel pattern of digested and electrophoretically separated eukaryotic chromosomal DNA upon hybridization with said oligonucleotide probes of the present invention.
  • the hypervariable probes of the present invention can be directly derived from eukaryo ⁇ tic genomes, can be of synthetic origin, i.e. obtained by DNA synthesis considering the chromosomal eukaryotic DNA se ⁇ quences identified by the present invention, or can be ob ⁇ tained by cloning any of the aforementioned DNA sequences or parts thereof.
  • said hy ⁇ pervariable probes are capable of hybridizing with any of the chromosomal eukaryotic DNA sequences shown in Figures 1- 6 or with any of their complementary DNA sequences.
  • Particu- larly preferred oligonucleotides of the present invention are underlined in these Figures.
  • said hypervariable probes are capable of hybridizing to eukaryotic DNA sequences which hybridize with any of the above-mentioned eukaryotic DNA sequences.
  • hybridization preferably refers to hybridization conditions for cloned probes under which the T m value is between T m -20 to T m -27'C, preferably T m -20 to T m -25*C (the preferred T m value for oligonucleotides is about T m -5 ⁇ C; see e.g. Suggs et al. (11)). More preferably, the term “hybridization” refers to stringent hybridization conditions. Such hybridization conditions are known to the person skilled in the art.
  • hypervariable DNA sequences are immediately adjacent to simple repeat DNA sequences.
  • simple repeats refers to short DNA motifs of up to 10 bases reiterated over and over in a head to tail fashion, i.e. "...CAC CAC CAC CAC".
  • simple repeats contain the sequence motif (CAC) 5 /(GTG) 5 in humans or (GGAT) 4 in fish or (GAA) 6 in plants.
  • the eukaryotic DNA sequences to which the hypervariable pro ⁇ bes of the present invention hybridize are obtainable from eukaryotic genomes by (partial) digestion of chromosomal eu ⁇ karyotic DNA with restriction enzymes, ligation of the frag ⁇ ments into appropriate vectors, establishing a genomic library in an appropriate host, screening the genomic lib ⁇ rary with oligomeric DNA probes derived from hypervariable eukaryotic DNA regions such as (CAC) 5 , and selecting the po ⁇ sitive clones. Individual clones are then tested for their informativity (heterozygosity rate) .
  • Respective cloned DNA fragments are subcloned in appropriate plasmid vectors and sequenced by established methods.
  • PCR semi-specific ligation-mediated polymerase chain reaction
  • adapter ligation after isola ⁇ tion of genomic DNA fragments using primers for the adapter and the simple repeats such as (CAC) 5 /(GTG) 5 can be used for generating the above-mentioned hypervariable DNA probes.
  • the invention further relates to the use of the above-men ⁇ tioned hypervariable probes as labelled probes in hybridiza ⁇ tion methods.
  • radioactive la ⁇ bels are radioactive la ⁇ bels.
  • said label is one of the conventional non-radioactive labels, such as digoxigenin, biotin or alkaline phosphatase.
  • hypervariable probes of the present invention can be used for the above-mentioned pur ⁇ pose irrespective of which of the two strands of the chromo ⁇ somal eukaryotic DNA they hybridize to because the relevant feature is the sequence specificity.
  • the present invention also relates to a kit fcr the specific detection of individual eukaryotic chromosomal loci, wherein this kit contains at least one of the hypervariable probes of the present invention.
  • the kit also contains other reagents which are conventionally used and applied in hybridization methods.
  • the figures show:
  • FIG. 1-6 Chromosomal eukaryotic DNA sequences to which the hypervariable probes of the present inven ⁇ tion hybridize.
  • Preferred subregions, i.e. re ⁇ gions representing preferred oligonucleotides of the present invention or their complemen ⁇ tary strands are underlined.
  • Figure 7 Mbol digested and electrophoretically separated DNA of 9 unrelated individuals probed with (a) Hzall, (b) Hza2, (c) Hza31 and (d) Hza4. In lane 3 (HZ4) less DNA was loaded. Molecular weight markers are indicated on the right in kb.
  • Figure 8 Chromosomal assignment of HZ1, HZ2, HZ3 and HZ4 using a panel of somatic cell hybrids (CH).
  • + human chromosome present in CH
  • (+) human chromosome present in ⁇ 10% of CH
  • Hybridization signals lacking in (+) and I CH were not scored as discordance.
  • the incon ⁇ sistencies in case of HZ4 (lacking signals in CH 11 and 14; additional signal in CH 7) were clarified by additional experiments.
  • Figure 9 Distribution of allele size, number and fre ⁇ quency of the hypervariable locus HZ3 in 17 eurasian population samples, Genomic DNA was restricted with Hinfl and hybridized to the Hza32 probe developed from the HZ3-locus.
  • the column base represents 0.1-kb intervals, the column height indicates the observed number [B] and the frequency [A] of each fragment class. In total 804 unrelated healthy individuals were tested.
  • the column base represents 0.1- kb intervals, the column height indicates the frequency of each fragment class.
  • Figure 11 Agarose gel electrophoresis of individual PCR reactions with one primer pair for each of the tetranucleotide hypervariable loci and simultaneous multiplex PCR's of all three loci. Note that the primers have been selected to allow the simultaneous analysis of the resulting PCR products. Additional bands of apparently higher molecular weight are explainable as partly single-stranded heterodimers between two different alleles originating from the same locus.
  • the hypervariable monolocus probes of the present invention identify essentially only 1 to 2 bands per individium. The actual number depends on the length differences between both hybridizing alleles.
  • the chromosomal eukaryotic DNA sequences of the present invention have been allocated to different human chromosomes. With a collection of the highly informative hypervariable monolocus probes of the present invention, the human genome can be mapped very efficiently.
  • the hypervariable monolocus probes of the invention for the first time permit the observation of these particular single chromosomal loci in a given individual. Thus, for the first time they permit a correlation between DNA sequences "located in the genomic desert" and for instance particular diseases.
  • DNA isolation, restriction enzyme digestion, electrophore- sis, Southern blotting or gel drying and probing with either cloned probes or oligonucleotides were carried out following standard protocols (4, 10, 13) .
  • final washing stringencies after hybridization of cloned probes were 0.1 x SSC at 68"C.
  • the oligonucleotide probes were labeled at the 5' end via a kinase reaction. Labeled oligonucleotides were separated on a denaturing polyacrylamide gel. 10 cpm were used per 1 ml of hybridization solution. Gel-hybridizations of oligo ⁇ nucleotide probes were carried out for 3h at T m -5 ⁇ C in the presence of 5 x SSPE at room temperature followed by a one minute lasting stringent wash at the respective hybridiza ⁇ tion temperature (19) . Exposure times were 4 days and 8 days with intensifying screens.
  • Plasmid DNA was obtained by the alkaline lysis method (13) followed by ion exchange chromatography (Quiagen, Diagen) . Double strand sequencing was done by the dideoxy chain termination procedure. Synthesis, deprotection, purification and labeling of oligonucleotides were performed essentially as described in (4) .
  • Example 1
  • the fragment was then digested with the restriction enzyme Pst I and the (CAC) ---posi ⁇ tive Pst I subfragment was identified.
  • the (CAC) 5 posi ⁇ tive Pst I subfragment was subsequently purified from LMP-agarose gels and further digested with Alu I to create a 601 bp repeat flanking subfragment. This Alu I subfragment was cloned into the plasmid pUC19 and used as probe in Southern hybridization experiments.
  • Xhol half sites were prepared by partially filling in Xhol generated, 5' protruding ends with a final concentration of ImM dTTP and ImM dCTP using 2U Klenow polymerase (New England Biolabs) at 37°C for 30 in in 50 mM Tris/HCl (pH 7.2), lOmM MgS0 4 and ImM DTT.
  • the linearized vector was dephos- phorylated in order to prevent self ligation of vector bearing unmodified Xhol sites. Before ligation the Mbol cut genomic DNA was also partially filled in with dGTP and dATP to produce sticky ends fitting to the Xhol half sites.
  • both oligonucleotides were annealed and ligated to EcoRI cut lambda ZAP II DNA in a thousand fold molar excess (16 ⁇ C/3h) .
  • the non-ligated adaptor was removed by gel filtration (Sephadex S200, Pharmacia) .
  • in vitro packaging phages were plated on XL-1 blue cells. Both libraries were screened with 32 p- labeled (CAC) 5 and from altogether 205,000 recombinant clones 6 (CAC) 5 /(GTG) 5 strongly positive ones were detected.
  • Competent JM 109 cells (recA, endA, gyrA96, thi, hsdR17, supE44, relAl) were transformed according to (15) . Several colonies from each time interval were isolated and their plasmid DNA analyzed.
  • oligonucleotide probes used in Examples 2, 4 and 5 are based on the sequences of Hzal, Hza3 and Hza4 and have the following sequences (or complementary sequences) :
  • Table I Heterozygosity rate (H) and mean allele frequency (q) of the hypervariable probes Hzal2, Hza2, Hza32 and Hza4.
  • the hypervariable probes for HZl, HZ2, HZ3 and HZ4 were chromosomally localized by probing DNA of somatic cell hybrids (Fig. 8) .
  • the short cloned repeat flanking probes generated weak hybridization signals and allowed only to assign HZ4 to human chromosome 22. This was confirmed by probing DNA of a menigneoma cell line lacking one chromosome 22 as revealed by scoring 30 identical etaphase plates (45XX, -22) .
  • the sensitivity could be improved by hybridizing the full length repeat containing clone in the presence of human competitor DNA allowing to map it to chromosome 8.
  • HZ3 and HZl amplified DNA containing several fragments of different sizes were subjected to electro- phoresis, blotted and challenged with internal oligo- nucleotide probes.
  • Bands of uniform size (184bp) allowed to map HZl to chromosome 11.
  • the data pertaining to HZ3 are initially more difficult to interpret.
  • the amplified region is also present in rodent DNA. Therefore, a principally identical banding pattern was obtained for each sample with one clearly predominant band of the expected size (324 bp) in several samples. The same quantitative differences were observed after hybridization with an internal oligo- nucleotide probe. Based on these quantitative differences HZ3 can be localized on chromosome 9.
  • a panel of more than 800 DNA samples extracted from unrela ⁇ ted individuals has been digested with the restriction enzyme Hinfl and hybridized with the locus specific oligonucleotide probe Hza31. All population samples ex ⁇ hibited a large number of alleles of different fragment lengths. The majority of probands showed two signal bands reflecting the high degree of heterozygosity.
  • Apparent homozygosity observed in a minority of the probands may be due to (i) either two fragments that cannot be resolved during the electrophoretic separation or (ii) the exclusion boundary of the gel system used causing DNA frag- ments smaller than 1.6 kb to run off the gel and hence escaping detection.
  • HZ4 specific alleles (Fig. 10) of the chromosome 9 show an abundance of fragments ranging between 1.5 kb and 4 kb of length. Similarities between Hinfl-digested (Fig. 9) and Mbol-digested DNA (Fig. 10) are due to the short distances between Hinfl- and Mbol-restric- tion sites flanking DNA fragments carrying variable numbers of tandemly organized (CAC) n /(GTG) n motifs.
  • CAC tandemly organized
  • More than 90% of the alleles of the HZ4 locus range from 0.9 kb to 5 kb. Although similarities to the HZl pattern cannot be neglected the distribution of the most frequent fragments is shifted to 2.5 kb to 3 kb.
  • Table II Degree of heterozygosity and homozygosity in three population samples from northern Germany, Assam, and northeastern Thailand.
  • Human chromosomal DNA was isolated from peripheral blood according to Miller et al. (16) by a salting out procedure. 50 - 1000 ng DNA were used for each PCR. The locus-specific primers (hypervariable probes, obtained substantially as described in Example 1) were synthesized.
  • PCR reactions were carried out according to the condi ⁇ tions proposed by the manufacturer of the Taq DNA polymerase (Perkin Elmer, Norwalk, CT, USA) at the following temperatures: denaturation at 94 ⁇ C for 30s, annealing at 51 ⁇ C for 30s and extension at 72 ⁇ C for 90s. 30 cycles were carried out in a reaction volume of 25 ⁇ l (2mM Mg ++ ) . Gel purification of the repeat containing fragments, radioactive labeling of the fragments, separation in 4% denaturing polyacrylamide gels and X-ray film exposure were performed as described by Roewer et al. (18) .

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Abstract

Sondes hypervariables capables de s'hybrider avec une séquence d'ADN chromosomique eukaryotique, ladite séquence d'ADN eukaryotique ayant les caractéristiques suivantes: a) elle ne se produit que sous la forme d'une seule copie par génome haploïde (monolocus); b) elle est située dans le chromosome immédiatement adjacent à une séquence d'ADN hypervariable; et c) elle est hétérozygote à au moins environ 70 %, et de préférence à 90 %. On peut utiliser ces sondes hypervariables comme sondes de détection de loci à exemplaire unique spécifique dans de l'ADN chromosomique eukaryotique.
PCT/EP1992/000269 1991-02-07 1992-02-07 Sondes hypervaribles a specificite monolocus WO1992013968A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5955276A (en) * 1994-11-28 1999-09-21 E.I. Du Pont De Nemours And Company Compound microsatellite primers for the detection of genetic polymorphisms

Citations (7)

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Publication number Priority date Publication date Assignee Title
EP0238329A2 (fr) * 1986-03-19 1987-09-23 Zeneca Limited Sondes génétiques
WO1989007658A1 (fr) * 1988-02-18 1989-08-24 University Of Utah Identification genetique avec des adn d'investigation ayant des sites repetitifs en tandem en quantite variable
EP0337625A2 (fr) * 1988-04-13 1989-10-18 Imperial Chemical Industries Plc Sondes
WO1990004040A1 (fr) * 1988-10-11 1990-04-19 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede pour l'analyse de polymorphismes longitudinaux dans des regions d'adn
EP0382261A2 (fr) * 1989-02-10 1990-08-16 Virginia Mason Research Center Sondes d'ADN contre les loci de VNTR
EP0390518A2 (fr) * 1989-03-30 1990-10-03 Lifecodes Corporation Compositions et procédés applicables à l'étude du dosage génétique
WO1991010748A1 (fr) * 1990-01-16 1991-07-25 Collaborative Research, Inc. Sequences d'adn provenant de loci genomiques humains specifiques utiles a l'identification d'individus

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238329A2 (fr) * 1986-03-19 1987-09-23 Zeneca Limited Sondes génétiques
WO1989007658A1 (fr) * 1988-02-18 1989-08-24 University Of Utah Identification genetique avec des adn d'investigation ayant des sites repetitifs en tandem en quantite variable
EP0337625A2 (fr) * 1988-04-13 1989-10-18 Imperial Chemical Industries Plc Sondes
WO1990004040A1 (fr) * 1988-10-11 1990-04-19 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede pour l'analyse de polymorphismes longitudinaux dans des regions d'adn
EP0382261A2 (fr) * 1989-02-10 1990-08-16 Virginia Mason Research Center Sondes d'ADN contre les loci de VNTR
EP0390518A2 (fr) * 1989-03-30 1990-10-03 Lifecodes Corporation Compositions et procédés applicables à l'étude du dosage génétique
WO1991010748A1 (fr) * 1990-01-16 1991-07-25 Collaborative Research, Inc. Sequences d'adn provenant de loci genomiques humains specifiques utiles a l'identification d'individus

Non-Patent Citations (1)

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Title
CHEMICAL ABSTRACTS, vol. 115, no. 5, 5 August 1991, Columbus, Ohio, US; abstract no. 43421Y, ZISCHLER,H.: 'OLIGONUCLEOTIDE FINGERPRINTING WITH (CAC)5:NON RADIOACTIVE IN-GEL HYBRIDIZATION AND ISOLATION OF INDIVIDUAL HYPERVARIABLE LOCI.' page 246 ;column L ; *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5955276A (en) * 1994-11-28 1999-09-21 E.I. Du Pont De Nemours And Company Compound microsatellite primers for the detection of genetic polymorphisms

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