WO1992013968A1 - Monolocus-specific hypervariable probes - Google Patents

Monolocus-specific hypervariable probes 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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French (fr)
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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/en

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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) .

Abstract

Hypervariable probes are disclosed which are capable of hybridizing with a eukaryotic chromosomal DNA sequence, said eukaryotic DNA sequence having the following features: a) it only occurs as a single copy per haploid genome (monolocus); b) it is located in the chromosome closely adjacent to a hypervariable DNA sequence; and c) it is at least about 70 %, preferably 90 % heterozygote. These hypervariable probes can be used as probes for the detection of specific single copy loci in eukaryotic chromosomal DNA.

Description

Monolocus-spβcific Hypervariable Probes
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 109 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 3500000000 (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 10108 A3 (cubic Angstrom) . More then 90 percent of the diploid cell nucleus with about 7 x 109 nucleotides is a "genetic desert", i.e. does not contain any sequence-dependent information. Thus, the genes appear as "small oases in the barren stretch of desert". Naturally, 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) . Thus, 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. However, about 30 percent of the genome con¬ sist of repetitive sequences which are very differently or¬ ganized with regard to copy number and structure. An inter¬ esting class of such repetitive sequences are the simple re- petitive elements which are composed of surprisingly often reiterated sequence motifs, e.g. ...gt gt gt gt gt gt gt... Some of these simple sequences are relatively evenly dis¬ tributed among the entire human genome. As they are located in the genetic desert, simple repetitive sequences are not translated into protein and do not contain sequence-specific information for the organism. These parts of the genome are thus particularly useful in methods for analysis of the ge¬ netic individualism, i.e. the genetic fingerprint. The prin¬ ciple of such methods was described for the first time in 1985 by Jeffreys et al (3) . A corresponding method of the present inventor is equally effective but can be carried out easier and faster and thus much more economical; see EP- A2 266 787.
It seems to be expedient to briefly summarize the steps com¬ prised in the above mentioned method: Preparation of the nuclear DNA of cells of particular tissues or secretions as blood, liver secretions, saliva, sperm, hair roots and other organs; cleavage of the DNA with particular restriction en¬ zymes; electrophoretic, length dependent separation of the resulting DNA fragments in a gel; fixing the DNA in or onto a solid matrix and separation of the double strand into the complementary single strands; hybridization with a probe which is complementary to the simple repetitive sequences; and preparation of the genetic fingerprint by using x-ray film in case of radioactively labelled probes (or chemo- luminescence) or by antibody-mediated enzymatic dye reactions. The probes are either chemically synthesized oligonucleotides labelled at their 5'-end with 32P-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). Depending on the multilocus oligonucleotide DNA probes and length variation of the hybridizing target DNA fragment, 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) . However, 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. On the other hand, 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) .
Thus, 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.
The solution of the above technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to 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) ;
(b) it is located in the chromosome closely adjacent to a hypervariable simple repeat DNA sequence; and
(c) it is at least about 70%, preferably 90% heterozygote.
Thus, the probes of the present invention are locus-spe¬ cific.
According to the present invention, the term "locus specific probe" or "monolocus 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. In this context, it has to be understood that 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. As regards human beings, the haploid genome comprises about 3.5 x 109 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.
Thus, the term hypervariable probe refers to the chromosomal eukaryotic DNA sequences as characterized in the present in¬ vention, fragments thereof and oligonucleotides derived the¬ refrom.
The term "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- gestion with enzymes recognizing 4 bases, such as Hinfl, Haelll, Alul or Mbol.
The term "hypervariable DNA sequence" refers to DNA se¬ quences that exceed the normal variability (polymorphism) by several orders of magnitude.
Furthermore, 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. In preferred embodiments of the present invention, 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.
It is once more emphasized that 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.
In a preferred embodiment of the present invention 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.
In another preferred embodiment of the present invention said hypervariable probes are capable of hybridizing to eukaryotic DNA sequences which hybridize with any of the above-mentioned eukaryotic DNA sequences.
The term "hybridization" as used herein preferably refers to hybridization conditions for cloned probes under which the Tm value is between Tm-20 to Tm-27'C, preferably Tm-20 to Tm-25*C (the preferred Tm value for oligonucleotides is about Tm-5βC; 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.
In another preferred embodiment of the present invention said hypervariable DNA sequences are immediately adjacent to simple repeat DNA sequences.
The term "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...". Preferably, such 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. Alternatively, semi-specific ligation-mediated polymerase chain reaction (PCR) including 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.
These DNA sequences and synthetically produced oligomers derived therefrom were tested as labelled monolocus-specific probes by hybridization with digested and electrophoreti- cally separated eukaryotic chromosomal DNA according to con¬ ventional methods which are for instance described in (9) or (10).
The invention further relates to the use of the above-men¬ tioned hypervariable probes as labelled probes in hybridiza¬ tion methods.
A preferred embodiment of such labels are radioactive la¬ bels. In another embodiment of the present invention said label is one of the conventional non-radioactive labels, such as digoxigenin, biotin or alkaline phosphatase.
It has to be understood that the 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. Optionally, the kit also contains other reagents which are conventionally used and applied in hybridization methods. The figures show:
Figures 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, I — presence of isozyme markers in the absence of cytogenetically identifiable chromosome (fragments) , n.d.= not tested.
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.
Figure 10: Distribution of allele size and frequency of the hypervariable loci HZ1, HZ3, and HZ4 in three populations of different ethnic origin: Germans, Assamese Hindus, and individuals from northeastern Thailand. Mbol digested high molecular weight DNA was hybridized consecu¬ tively to the locus specific oligonucleotide probes Hzal2 (n = 145 probands) , Hza32 (n = 145 individuals) , and Hza41 (n = 128 subjects) respectively. 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.
As opposed to the complex fingerprint obtained with the multilocus probes of the prior art, 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.
The examples illustrate the invention.
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) .
Unless otherwise indicated, 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 Tm-5βC 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
Preparation of locus specific hypervariable probes.
(A) 10 μg human male placental DNA was partially digested with the restriction enzyme Sau3A I, ligated into the arms of the phage vector Charon 21A and packaged in vitro following standard protocols (10) . Phages were absorbed on E. coli LE 392 bacteria. 1 x 105 pfu were plated and screened with the oligomeric DNA probe (CAC)5 labelled with 3 P-ATP. 24 randomly selected plaques showing positive signals were isolated and further purified. From small scale liquid cultures phage DNA was prepared and the DNA was digested to completion with Sau3A I. The clone bearing the largest (about 3.5 kb) fragment giving a positive signal with the probe (CAC)5 was further analyzed. 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.
(B) DNA from three unrelated persons was pooled, digested to completion with the restriction enzyme Mbol and run on a preparative agarose gel. The gel slice encompassing the 3-10kb region was isolated and DNA was electro-eluted. After ion exchange purification (Quiagen column, Diagen) the DNA was ligated into the lambda ZAP II vector (Stratagene) . Because this vector lacks a unique BamHI site, two alternative approaches were chosen:
(i) 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 MgS04 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. After overnight ligation at 16 "C the DNA was packaged in vitro (Gigapack Gold, Stratagene) and plated on XL- 1 blue cells (recAl, lac, endAl, gyr96, thi, hsdR17, supE44, relAl) . (ii) The alternative approach included EcoRI-BamHI con¬ version adaptors basically following the methodology described in (14) . Two oligonu¬ cleotides were synthesized: A 5'AATTCGAACCCCTTCG and B 5'GATCCGAAGGGGTTCG. After kinasing A, both oligonucleotides were annealed and ligated to EcoRI cut lambda ZAP II DNA in a thousand fold molar excess (16βC/3h) . Before ligating the genomic DNA to the vector, the non-ligated adaptor was removed by gel filtration (Sephadex S200, Pharmacia) . After in vitro packaging phages were plated on XL-1 blue cells. Both libraries were screened with 32p- labeled (CAC)5 and from altogether 205,000 recombinant clones 6 (CAC)5/(GTG)5 strongly positive ones were detected. The conversion of the (CAC)5/(GTG)5 positive lambda ZAP II clones to pBluescript phagemids was done according to the in vivo excision protocol supplied by the manufacturer (Stratagene) . Unidirectional deletions in the plasmid inserts were generated by exonuclease III/mung bean nuclease treatment following the supplier's protocol (Stratagene) . Before self ligation the reaction mixtures from each incubation interval were electrophoresed on a preparative LMP agarose gel and the major bands were excised. Thereafter DNA was purified by absorption onto glass milk (Geneclean, BIO 101) and religated overnight. 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.
Single strand sequences of the clones were obtained by plasmid sequencing using the dideoxy chain termination method. The resulting sequences of the clones Hzal to Hza6 are depicted in Figures 1 to 6.
The 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) :
Hzall 5'-ATGTAGGCTTAGATTACATAGG-3' Hzal2 5'-AATTCTGGACAATGAGTAG-3' Hza31 5'-AGAGATTTAATTTCACTGAGCA-3' Hza32 5'-GATCTGAATCTTCAATTTGAC-3'
Example 2
Heterozygosity rates and allele size distributions
In order to initially estimate the heterozygosity rates of the isolated probes for the respective loci HZ1, HZ2, HZ3 and HZ4, DNA from 9 unrelated Caucasians was digested with the restriction enzyme Mbol and challenged with the repeat flanking subclones. Figure 7 shows the hybridization pat¬ terns for the probes Hzall, Hza2, Hza31 and Hza4. Already at first sight all these probes clearly display a high degree of polymorphism, with most of the individuals being heterozygous at the respective loci. The samples showing only one allele are either (i) homozygous at the respective locus, (ii) small lengths differences could not be resolved or (iii) the second signal band ran off the gel. The final conclusion is most likely because of the independently established distribution of the allele lengths including data from short run gels. For minimum estimates of heterozygosity, all persons exhibiting one or no band were regarded here as homozygous. The preliminary minimum estimates of heterozygosity rates (H) and mean allele frequencies (9) are based on the allele sharing values (s) and calculated as proposed by (16) with H = (1-s)1'2 and q = 1-H. Due to the limited number of samples and limited electrophoretical resolution the H values are grossly underestimated (Table I) .
Table I: Heterozygosity rate (H) and mean allele frequency (q) of the hypervariable probes Hzal2, Hza2, Hza32 and Hza4.
Figure imgf000016_0001
The respective loci were also examined in 3 Caucasian fami¬ lies (8 children) and all are inherited according to the Mendelian rules.
Example 3
Chromosomal Assignment
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) . Fine mapping data obtained from challenging hybrid cell lines containing different chromosome 22 frag¬ ments allowed to map Hza4 to 22ql2.1 - 22ql2.1. For HZ2 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.
In the case of 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.
Example 4
Frequency distribution of alleles of the hypervariable locus HZ3 in 17 eurasian populations after Hinfl digestion.
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.
In general, all 17 population samples show approximately the same modal distribution and abundant representation of al¬ leles in the molecular range of 1.6 kb to 5 kb which is obviously characteristic for the locus HZ3 located on chromosome 9 (Fig. 9) . In this distribution fragments ranging between 2.5 kb and 3.5 kb show maximal frequencies. In the distribution profile alleles exhibiting a size between 5 kb and 11 kb show significantly lower frequencies. A considerable larger fragment of 17 kb length could be visualized only once in a subject from Northeastern- Thailand.
Example 5
Frequency distribution of alleles of the hypervariable loci HZl. HZ3 and HZ4 in Eurasian populations after Mbol digestion
DNA samples from German, Assamese Hindus, and probands from northeastern Thailand (Thais) were restricted with the restriction enzyme Mbol and hybridized with the locus specific oligonucleotide probes Hzall, Hza31 and Hza41. As documented in Table 2 probands of the Eurasian population samples showed considerably high degrees of heterozygosity at the hypervariable loci HZl, HZ3 and HZ4. The frequency distribution of HZl alleles of chromosome 11 is documented in Figure 10 covering a total range between 0.9 kb and 11.2 kb. Although a predominant representation of DNA fragments is visible between 1 kb and 5 kb, the profile does not show a distinct peak.
The focal distribution of 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.
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.
Figure imgf000019_0001
Amounts of 10 μg of DNA were digested with the restriction enzyme Mbol. Following size fractionation, alleles of the hypervariable loci HZl, HZ3 and HZ4 were visualized.
* These individuals are not necessarily true homozygotes, since small scale differences cannot be detected by con¬ ventional agarose gel electrophoresis. In addition though it is less likely due to the exclusion boundary of 0.5 kb smaller DNA fragments might have run off the gel. Example 6
Multilocus PCR of three loci on the human Y chromosome and chromosome 12 using hypervariable probes as primers
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.
LOCUS 27H39: H39.1 5'-CTACTGAGTTTCTGTTATAGT-3' H39.2 5'-ATGGCATGTAGTGAGGACA-3'
5'-TCATTTAAGCATTTGAGGGAA-3' 5'-AGACTTCAAAACAGACACTT-3'
5'-GCAACAGTTTATGCTAAAGC-3'
Figure imgf000020_0001
5'-GCCTATTCAGTTCAAATCTA-3'
The 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 94βC for 30s, annealing at 51βC for 30s and extension at 72βC for 90s. 30 cycles were carried out in a reaction volume of 25μl (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) .
As shown in Figure 11, the informativeness is increased even though only minute amounts of material is available. Literature:
(1) Ali et al., Hum. Genet. 74 (1986), 239-243.
(2) Jeffreys et al., Nature 316 (1985), 67-73.
(3) Jeffreys et al., Nature 316 (1985), 76-79.
(4) Schafer et al., Electrophoresis 9 (1988), 369-374.
(6) Pόche et al.. Adv. Forensic Haemogenet. 3 (1990), 14-16.
(7) Georges et al., Geno ics 6 (1990), 461-474.
(8) Weising et al., Nucl. Acids Res. 17 (1989), 10128.
(9) Zischler et al., Nucl. Acids Res. 17 (1989), 4411.
(10) Maniatis et al., "Molecular Cloning, A Laboratory Ma¬ nual", Cold Spring Harbor Laboratory, N. Y. , 1982.
(11) Suggs et al. , Proc. Natl. Acad. Sci. USA 80 (1983), 3651-3655.
(12) Zischler et al., Hum. Genet. 82 (1989), 227-233.
(13) Sambrook et al., "Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Laboratory, N.Y., 1989.
(14) Stover et al.. Anal. Biochem. 163 (1987), 398-407.
(15) Perbal, "A practical guide to molecular cloning," Wiley & Sons, New York 1988.
(16) Way et al., Ann. Hum. Gent. 51 (1987), 269-288.
(17) Miller et al., Nucl. Acids Res. 16 (1988), 1215.
(18) Roewer et al., Hum. Genet, (in press).
(19) Thein and Wallace, "The use of synthetic oligo¬ nucleotides as specific hybridization probes in the diagnosis of genetic disorders," in K.E. Davis (ed.): Human genetic diseases; a practical approach, pp. 33- 50 IRL Press, Oxford, 1986.

Claims

1. A hypervariable probe 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 (monolocus) ;
(b) it is located in the chromosome closely adjacent to a hypervariable simple repeat DNA sequence; and
(c) it is at least about 70%, preferably 90% heterozy- gote.
2. The hypervariable probe according to claim 1, said eukaryotic DNA sequence having a DNA sequence as de¬ picted in any of Figures 1 to 6 or a part thereof or a complementary DNA sequence thereof.
3. The hypervariable probe according to claim 2 being any of the oligonucleotides underlined in Figure 1 or 3 or a complementary oligonucleotide thereof.
4. The hypervariable probe according to claim 1, said euka¬ ryotic DNA sequence being a DNA sequence hybridizing to a eukaryotic DNA sequence as given in claim 2 and displaying features (a) to (c) as recited in claim 1.
5. The hypervariable probe according to claim 1 or 4, said hypervariable DNA sequence being immediately adjacent to simple repeats.
6. The hypervariable probe according to any one of claims 1 to 5, said eukaryotic DNA sequence being obtainable from eukaryotic genomes by (partial) digestion of chromosomal eukaryotic DNA with restriction enzymes, ligation of the fragments into appropriate vectors, establishing a ge¬ nomic library in an appropriate host, screening the ge- nomic library with oligomeric DNA probes derived from hypervariable eukaryotic DNA regions such as (CAC)5, and selecting the positive clone.
7. Use of a hypervariable probe according to any one of claims 1 to 6 as a labelled probe in hybridization me¬ thods.
8. A kit for the specific detection of eukaryotic chromo¬ somal loci, said kit containing at least one hyper¬ variable probe according to any one of claims 1 to 6.
PCT/EP1992/000269 1991-02-07 1992-02-07 Monolocus-specific hypervariable probes WO1992013968A1 (en)

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