WO1990002818A1 - Probes for the detection of rflp in eucaryotic genomes - Google Patents

Abstract

A method for obtaining polynucleotide sequences which are useful for detecting Variable Tandem Repeat polymorphism at multiple genetic loci and other genetic analyses under high stringency conditions (hence, with high specificity) is herein disclosed. Also disclosed are polynucleotide sequences and other compositions useful for DNA polymorphism and other genetic analyses.

Description

DESCRIPTION

Pro_bes for t_he detection of RFLP in eucaryotic genomes.

FIELD OF THE INVENTION

This invention relates to the field of molecular genetics. More specifically, this invention relates to polynucleotides useful for nucleic acid hybridizations, methods for producing these polynucleotides, and methods for applying these polynucleotides in genetic analysis.

BACKGROUND OF THE INVENTION Double stranded DNA is the most common form of depository of genetic information of organisms. Double stranded DNA has two complementary strands. Each strand is a polynucleotide sequence and the base sequences on the two complementary strands form Watson-Crick base pairs. The duplex structure of DNA can be disrupted in a number of ways, for example, by heating a duplex DNA solution in a 0.1 M NaCl to 100°C for a few minutes. At this temperature, the two strands of duplex DNA separate. If the solution is gradually cooled, the two strands of duplex DNA can re-associate to reform the duplex structure.

The process of duplex formation from complementary polynucleotide or oligonucleotide sequences has been used advantageously for genetic analysis. Typically, a labeled polynucleotide or oligonucleotide sequence is used in a reassociation process whereby it forms a duplex structure with a substantially complementary sequence from a genetic source of interest. Because the labeled polynucleotide or oligonucleotide sequence is normally, though not necessarily, obtained from a source other than the source of interest, the process of association between complementary sequences has been known as nucleic acid hybridization, or just hybridization for short. The associational event provides genetic information about th source of interest through detection of the label on th labeled polynucleotide or oligonucleotide sequence. Fo this reason, the labeled polynucleotide or oligonucleotid sequence is called a probe. The label can be any suitabl signal-generating moiety, and many such moieties are wel known in the art.

Nucleic acid hybridization has been successfull applied in the study of DNA structure, gene purification, gene localization, the establishment of paternity and othe familial relationship, genetic identity for forensi purposes, genetic identity of transplants, and detectio and diagnosis of diseases and genetic traits.

One very powerful technique in the application o nucleic acid hybridization involves the fractionation o the complex genetic material to be analyzed prior t hybridization. E.M. Southern's procedure is the mos celebrated and the most widely used of this genus. Se Southern, J. Mol. Biol. 98: 503-517 (1975). Such a geneti analysis can reveal not only the presence or absence o complementary target nucleic acid sequences, but also th size of the restriction fragment(s) containing the targe sequence. Genetic variations within a species may b reflected by variations among individuals in the size o the restriction fragments containing a particular targe sequence. Conversely, genetic relatedness of a group o individuals may be reflected by a deviation from rando variations that exist among unrelated individuals. Thi aspect of genetic analysis has been called Restrictio Fragment Length Polymorphism (RFLP).

"Single-copy" DNA probes have been used in thi approach with success. For example, certain geneti traits and disease states have been identified this way. See Gusella et al., Nature, 306: 234-238 (1983); Orkin, Cell 47:845-850 (1986). The genetic information which can be adduced usin "single-copy" DNA probes depends on the number of probe used, the number of genetic loci each probe is capable o detecting, the heterozygosities and the allele frequency o the relevant genetic loci. To date, "single-copy" DN sequences are known to detect only a single locus pe sequence. Moreover, heterozygosity of DNA in highe organism is low. In man, it- is about 0.001 per base pair. Finally, most polymorphic states detected are onl dimorphic (i.e. there are only two representational states: absence or presence of a relevant restriction site on the restriction fragment in question). As is often the case, critical individuals in a genetic analysis are homozygous, and the genetic analysis may be uninformative. Genetic analysis in higher organisms has been simplified considerably by the availability of probes for hypervariable regions of genomic DNA. These hypervariable regions show multi-allelic variation and high heterozygosities. These regions also appear to be widely interspersed within the genome. In each case, the hypervariable region comprises a variable number of tandem repeats of a short sequence (thus, Variable Tandem Repeats or VTR), and polymorphism results from allelic differences in the number of repeats at a given locus. This type of polymorphism, a subclass of RFLP has been called VTR Polymorphism. It is believed that the variation in repeat number arises by mitotic or meiotic unequal exchanges or by DNA "slippage" during replication. Therefore, if genomic DNA is digested with a restriction endonuclease which does not cut within the repeat unit, and if a genetic locus encompasses a variable tandem repeat or VTR, allelic markers would exist for that locus. (It should be noted that the so-called repeat unit is a hypothetical consensus sequence, and any actual VTR sequence in the genome is really a string of short "core" sequences, each of which is very highly homologous, but usually not identical to th consensus sequence. Indeed, a "core" sequence may diffe in length from the consensus sequence. The consensu sequence is derived from examining and "averaging" over large number of "core" sequences. A "core" sequence i typically at least 70%, but often more than 70%, homologou to the consensus sequence. )

Jarman et al. have described a hypervariable region o DNA located 8-kb downstream of the human alpha globi complex. The EMBO J. 5: 1857-63 (1986). Thi hypervariable region is composed of an array of imperfec 17-bp tandem repeats, the number of which differ considerably (70-450) from one allele to another. Thus, this locus is highly polymorphic. Genetic polymorphis which reflects variations in the number of such tande among individuals has been called Variable Tandem Repea Length Polymorphism.

The VTR described by Jarman et al., supra, cross hybridizes with other hypervariable genetic loci at lo stringency. Thus a polynucleotide probe prepared from thi region is potentially a very powerful probe, capable o probing many genetic loci in a single try.

A typical RFLP analysis involves digesting targe genomic DNA with a restriction endonuclease, separating th digested DNA by gel electrophoresis, transferring th fractionated DNA in a denatured state to a binding surface, hybridizing the transferred DNA with a suitable probe, detecting the signals generated by the probe molecule which have become ^"hybridized to the target DNA. Th pattern of the signals generated would provide informatio about the target DNA. The pattern of signals can also b stored for later use, for instance, to determine or confir an individual's identification (i.e., the pattern would b the individual's genetic fingerprint) . More commonly, two or more target DNA's are processed for RFLP analysis. Depending on the sources of the target DNA, the information generated by comparison of th patterns can be used immediately as in the case of genetic identity (e.g., identification of a suspect of a crime), or in the case where a high degree of genetic relatedness is present (e.g., paternity testing, sib analysis and the like). In other cases, the information derived from pattern comparison may form a part of a larger information-gathering effort. Pedigree analysis of distant relatives and correlation of a gene of genotype with a trait or medical condition are but two examples.

However the RFLP analysis is to be used, the pattern of signals is controlled in large part by the probe or probes used in the analysis. A polynucleotide probe may be useful for any of a number of features.

First, a probe may be able to detect polymorphism at a locus that other probes cannot detect. The locus may be particularly useful for genetic analysis in the general population because it has many evenly distributed alleles. Alternatively, the locus may be particularly useful for genetic analysis in a highly restricted segment of the population because it has a rare allele.

Second, a probe may be able to detect many loci simultaneously and unambiguously when a particular restriction endonuclease is used to digest the target DNA. In this connection, it is useful to note that certain restriction endonucleases may be preferred because of the history of the target DNA samples, e.g. forensic samples which has been exposed to the elements for an extended period of time.

Third, probes are often used in combination simultaneously because their resolving power may be compounded. Compounding is obtained when the signals produced by the several probes do not overlap and permit unambiguous assignment of each (or substantially each) signal to an allele of a locus." See, e.g., "Th Application Of DNA-Print For The Estimation Of Paternity", Baird et al. in Advances in Forensic Haemogenetics 2: 354 358, Springer-Verlag, New York (1987). How RFLP phenotypes can be practically applied fo paternity and forensic determinations have been discusse in Baird et al., supra; Baird et al. (II), "The Applicatio Of DNA-PRINT™ For Identification From Forensic Biologica Materials", in Adv. in Forensic Haemogenetics 2: 396-402, Springer-Verlag, New York (1987); and Baird et al. (Ill), Am. J. Hum. Genet. 39:489-501 (1986) and citations therein These papers are hereby incorporated by reference.

Reference has been made earlier in the instan disclosure that hybrid formation can take place even wher there is a certain degree of mismatch between a probe an its substantially complementary target sequence'. Thi process is particularly important for the utility o multilocus probes. Such probes generally form well-matche hybrids with target sequences which originate from the sam genetic locus as the probe, but they form less well-matche "cross-hybrids" with target sequences from other loci. A a result, the loci that can be analyzed with a given prob may vary significantly with the reaction (association an washing) conditions of the hybridization test. Thus, man loci are detectable under low stringency conditions, bu only a single locus is detectable under high stringenc conditions. Therefore, a polynucleotide sequence which i capable of probing multiple polymorphic loci even unde high stringency conditions represent an additional bonus. Strictly speaking, stringency of conditions has tw components: conditions which govern formation of th hybrids and conditions which govern the stability of (th duplex structure of) the hybrids. Typically, however, hybridization test is performed at low (or relaxed conditions during the hybrid formation phase to speed u the process of association. Therefore, for the purpose o this application, stringency of conditions refer solely t conditions which govern the stability of the hybrids. (O course, if a hybrid is not stable under a given set o conditions, it would not be formed in the first place unde those conditions. )

The factors which govern the stability of a hybrid are many, including, but not limited to the temperature, the ionic strength, the molecular species of the salts used, the degree of modification or elimination of bases on a polynucleotide sequence, the degree and nature of mismatch, and the length and type of polynucleotides sequence. Variation in one factor may be compensated or aggravated by variations in other factors. These and other relevant facts are well known to a person of ordinary skill in the art of molecular genetics.

For the purpose of this invention, low stringency conditions mean an aqueous environment containing about 2X SSC at about 50-65 C, or the equivalents thereof; and high stringency conditions mean an aqueous environment containing about 0.1X SSC or less at about 65 C, or the equivalents thereof. [For formulation of IX SSC, see Example 3 in Section 6 below] .

For the purpose of this invention, a "discrete poly- nucleotide sequence or subsequence" means a polynucleotide sequence or subsequence of greater than 15 nucleotides, but preferably greater than 50 nucleotides, and very preferably greater than 100 nucleotides; and a polynucleotide means a chain of about 15 nucleotides or more, and embraces the upper range of what sometimes passes as oligonucleotides.

Many "single copy" DNA probes are known in the art.

These probes do not relate closely to the present invention because their utility is generally ( 1 ) limited to providing genetic information at a single locus; and (2) limited to detecting polymorphism caused by alteration of a restriction site in the neighborhood of the target genomic sequences. The polymorphic probes of the present invention are of the VTR type and do not suffer from these limitations.

Polymorphic probes of the VTR type have also been described. However, the hybrids formed between a VTR probe and its target genomic sequences tend to be stable only under low to moderate stringency conditions, except for hybrids between the probe and target sequences from a single genetic locus. See Nakamura et al. (I), Science 235: 1616-1622 (1987); Jeffreys et al., Nature 314:67-73 (1985). The exceptional hybrids are stable even under high stringency conditions, possible reflecting the fact that the probes originated from this locus.

Sometimes a genetic locus detectable with a VTR type probe may be very large, spanning several hundred kilobases. In a restriction fragment length polymorphism analysis of such a large locus, a VTR can yield many polymorphic bands under high stringency conditions. However, the information which can be derived from such an analysis remains confined to the one locus. In fact, VTR probes for loci of this kind have disadvantages. First, recombination within the large locus (which is expected to be more frequent than a similar but smaller locus) can complicate data analysis. Second, to obtain more extensive information than is obtainable from only a single locus, two or more probes are preferably used in combination. The multiplicity of non-informational bands from the large locus may obscure bands detected by other probes used in combination, thereby making data analysis very difficult. The alternative to using probes in combination would be more costly multiple analyses of restriction fragment length polymorphism. Therefore, the difference between a probe for a large single locus and a probe for multipl loci is substantive, and not merely semantic.

BRIEF SUMMARY OF THE INVENTION The subject invention concerns the following:

(1) polynucleotide sequences useful for detectin polymorphism in a species of organism of interest, or subpopulation thereof;

(2) a polynucleotide sequence useful for detectin polymorphism at multiple genetic loci, and characterized b its ability to form hybrids with restriction fragments of DNA, of 7.1, 6.2, 4.4, 4.2, 4.1, 3.7, 3.6, 2.6 and 2.2 kilobases, produced by Pstl digestion of genomic DNA extracted from K562 cells; (3) a polynucleotide sequence useful for detecting polymorphism at multiple genetic loci and capable of forming hybrids with genomic DNA fragments produced by complete digestion of Caucasoid, American Black or Hispanic genomic DNA with the restriction endonuclease Pstl of approximate allelic lengths and allelic frequencies as given in Table 2;

(4) a polynucleotide sequence useful for detecting polymorphism at multiple genetic loci with high specificity, i.e., the polynucleotide sequence forms hybrids with genomic sequences at multiple genetic loci which remain stable even under high stringency conditions;

( 5 ) a method for determining or obtaining a polynucleotide sequence useful for detecting polymorphism at multiple genetic loci with high specificity; (6) the use of the above-described polynucleotide sequence as a probe for polymorphism;

(7) the use of the above-described polynucleotide sequence as a probe for polymorphism at multiple genetic loci; (8) a method of genetic analysis comprising: ( a) digesting a DNA sample with a restrictio endonuclease ;

(b) separating the DNA restriction fragment according to size by electrophoresis; (c) transferring the separated DNA to a bindin surface;

(d) hybridizing the transferred DNA with polynucleotide probe labeled with a signal generating moiety, wherein the polynucleotid probe is a polynucleotide probe of the presen invention; and

(e) detecting the signal generated; whereby th pattern of signals generated provides informatio about the composition of the DNA sample; and (9) recombinant vectors and cells useful for producin polynucleotides of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the electrophoretic pattern o restriction fragments of DNA's from recombinant lambd phage 19 which was digested with EcoRI (Figure 1A), an the results of a Southern hybridization blot when the DN fragments were transferred and probed with oligo-5' CCCCCCGTGTCGCTGTT-3' (Figure IB). Figure 2 shows the results of 2 hybridization blots

Pstl digested human genomic DNA from a group of unrelated individuals were probed with either DNA fro Bands C of Figure 1A (Figure 2A), or DNA from Band D o Figure 1A (Figure 2B). Figure 3 shows the results of a hybridization blo where a panel of Pstl digested human genomic DNAs from fiv unrelated individuals was hybridized with either the inser of pAC329 (Figure 3A), or the insert of pAC344 (Figure 3B)

Figure 4 shows the electrophoretic patterns of th Rsal digests of the inserts of pAC329 and pAC344. Figure 5 shows the results of a hybridization blo where Pstl digested human genomic DNA was probed with Rsal restriction fragments of the inserts of pAC329 and pAC344. Figures 5A, 5B, 5C and 5D shows the results when Band 1 , 2, 3 or 4 of Figure 4 respectively was used as probe. Figure 6 shows the restriction map of pAC365. Figure 7 shows the results of a hybridization blot where Pstl digested human genomic DNA was probed with the insert of pAC365. Figure 8 shows the electrophoretic pattern of the restriction fragments produced by digesting the 1.35 kbp human insert of pAC365 with BstNI.

Figure 9 shows the results of a hybridization blot where Pstl. digested human genomic DNA was probed with the EcoRI-Pstl (Figure 9A) or the EcoRI-NcoI (Figure 9B) subfragment of the 1.35 kbp .human insert of pAC365.

Figure 10 shows the results of hybridization blots where Pstl digested human genomic DNA was hybridized with the insert of pAC365 under (1) low associational stringency and low stringency wash (Figure 10A); (2) low associational stringency and high stringency wash (Figure 10B); and (3) high associational stringency and high stringency wash (Figure 10C).

Figure 11 shows the results of hybridization blots where Pstl digested human genomic DNA was probed with the

1.35 kbp human insert of pAC256, and washed under either low stringency conditions (Figure 11A), or high stringency conditions (Figure 11B).

Figure 12 shows the comparative banding patterns in hybridization blots where Pstl digested human genomic DNA was probed with either "Band C DNA" (Figure 12A), or with 19-MSP (Figure 12B).

Figure 13 shows the results of a hybridization blot where Pstl digested genomic DNAs from a family spanning three generations were probed with the 1.35 kbp human insert of pAC365.

Figure 14 shows the allelic distributions of genetic loci detectable by pAC365 in American Blacks (Figure 14A), Caucasoids (Figure 14B), and Hispanics (Figure 14C).

Figure 15 shows the results of hybridization blots for genetic identification purposes. pAC365 insert was used as probe in a paternity test (Figure 15A), and a forensic test (Figure 15B). Figure 16 shows the relationships among the human sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION One embodiment of the instant invention is a method to obtain polynucleotide sequences useful for detecting polymorphism in a species, or a subpopulation thereof. A library of genomic DNA digested with one or more restriction endonucleases and cloned in a suitable recombinant vector is screened with a polynucleotide probe which comprises a string of "core" sequences (hereinafter "screening probe"). This string of "core" sequences can, but need not be, a monomer, an oligomer or a polymer or a mixture of oligomers and polymers of a consensus sequence or "core" sequence of a VTR. Preferably, the screening probe is a mixture of oligomers of a consensus sequence, because a short consensus sequence can be easily synthesized chemically in large amounts and ligated to form a mixture of oligomers. In a preferred embodiment, the consensus sequence is 5'-CCCCCCGTGTCGCTGTT-3' . For the purpose of generating a genomic library, it is preferred that the restriction endonuclease digestion of genomic DNA be incomplete. One reason is that many genomic VTR sequences may otherwise evade detection. This would be so if the relevant restriction endonuclease cuts within the VTR sequences, and the bulk of the VTR sequences will be in relatively small pieces. The smaller the pieces, th greater the number of recombinant molecules which must b studied so that the human genome will be covered. For th same reason, it is preferred that a recombinant vecto which can accommodate large DNA insert be used. Finally, where the recombinant vector has a restricted clonin range, incomplete digestion of the genomic DNA would als tend to avoid under-representation in the library of completely digested products which are smaller than the preferred cloning sizes.

The recombinants which react positively with the screening probe in a hybridization test (hereinafter

"positive recombinants") are selected for further examination. In a preferred embodiment, the recombinants are bacteriophages. The standard method of "phage lifts" can be used to identify the recombinants containing DNA inserts which hybridizes to the probe. See Maniatis et al.. Molecular Cloning: A Laboratory Manual, Cold Spring

Harbor Laboratories, Cold Spring Harbor, New York, (1982). Briefly, a portion of a phage plaque is transferred to a nylon membrane where the DNA of the phage is immobilized and probed. Many plaques can be transferred in a single lift; moreover, the position of a plaque on the growth plate is in a one-to-one correspondence with the position on the membrane, thus permitting identification of the plaques which give rise to positive results in a hybridization test with the probe. Obviously, many variations of this basic technique can be designed with other cloning and/or transfer and/or identification systems.

Once the positive recombinants have been identified, they can be subjected to tests which prove or disprove their utility. They are used as probes in hybridization tests against genomic sequences of a species of organism of interest, or a relevant subpopulation thereof. While the present invention broadly encompass eukaryotic organisms, one of the more commercially significant use is that of probing mammalian genomes, particularly, the human genome. In any case, it is very highly preferred that the probe sequences of the present invention be derived from the same species of organisms as the genetic materials which are to be tested in a hybridization test. Thus, for applications of human genetic analysis, the starting library should preferably be a human genomic library. To avoid verbosity, the embodiments of this invention are described as if they apply to humans specifically. The present invention is not so limited, and is to be construed to be applicable generally to mammals and other eukaryotes.

The useful positive recombinants are those which can detect Variable Tandem Repeat Length Polymorphism i humans, at multiple loci, and under high stringenc conditions. Genomic DNAs from family members are separately digested with a restriction endonuclease, the digests are separately subjected to size fractionation by, for example, electrophoresis, and the fractionated restriction fragments are prepared for hybridization i any standard method. Positive recombinants or the huma sequences or discrete polynucleotide subsequences inserted therein (jointly and severally "test sequences") are used to probe the restriction digests. Preferably, a singl test sequence is used at a time. However, several sequences can be grouped together in preliminary tests to determine whether the group as a whole contains any useful sequences. The hybridization "banding" pattern for eac individual member is determined. In particular, the sizes of restriction fragments which hybridize to the tes sequences are determined. The segregation scheme of eac band within a family or, more commonly, a number o families will inform as to the nature of the genetic locus (loci) being detected. The nature of a genetic locu includes, but is not limited to, the following: 1. Mendelian or non-Mendelian segregation; 2. phenotype an frequency of alleles (reflected by the size of restrictio fragments produced by the restriction endonuclease used t digest the genomic DNA, and the frequency of occurrence i a population); 3. linkage to another locus (reflected b co-segregation with other bands). If the test sequenc detects multiple bands which segregate independently, it i capable of detecting multiple genetic loci. If tes sequence detects bands of a locus which bands represen different-sized fragments among different individuals, it is capable of detecting polymorphism in that polymorphic locus. The determination of the nature of the locus (loci) detected by a test sequence from the segregation scheme is a straight forward application of classical genetics, and is well within the command of a person of ordinary skill in the art of molecular genetics. The size of families, and the number of families needed to provide sufficient information to work out the segregation scheme would vary with the number of genetic loci being detected by the test sequence, the number of alleles in these loci, and the frequency of each allele. An ordinarily skilled artisan would also know how to determine the number and size of families to be studied.

Another embodiment of the instant invention is the test sequences which can detect, polymorphism in an species of organism of interest, or a subpopulation thereof (hereinafter"useful test sequence"). In another embodiment of the instant invention, useful test sequences are cloned in recombinant vectors. In another embodiment, the recombinant vectors comprising the useful test sequences are harbored in a cell. Molecular cloning and transformation methods are well known in the art. In a preferred embodiment, the useful test sequences are such that the hybrids formed between the test sequence and genomic sequences from at least two genetic loci are stably associated under high stringency conditions. Because the segregation scheme is both lengthy and expensive to workout, it is sometimes preferably to defer the study of segregation until a test sequence has been better characterized. Thus, it may be preferable to modify the method described hereinabove, namely, to use instead of genomic DNAs from members of families, merely genomic DNAs from random, unrelated individuals. If the banding pattern appear to vary from individual to individual, the test sequence is presumptively treated as being useful as a polymorphic probe. If the number of bands detected in eac of several individuals is significantly greater than one o two, the test sequence is presumptively treated as bein useful as a multi-locus probe.

The presumptively useful test sequence is analyzed, and less desirable sequence(s) are removed to produce a improved test sequence. For example, a test sequence ma comprise a subsequence which is polymorphic as well as subsequence which is non-polymorphic in the relevan population. The presence of non-polymorphic bands yields no useful genetic information about a human individual, but can interfere with genetic analysis by, for example, obscuring a partially or totally informational ban detected by a second probe used in combination with th test sequence. Another example is where the test sequenc comprises a subsequence which is a highly repetitiv sequence in the human genome. An example of a highl repetitive sequence is the "Alu sequence". See Houck e al., J. Mol. Biol. 132: 289 -306 (1979). Presence of suc highly repetitive sequences in a probe often cause a hig non-informational background signal in a hybridizatio blot. This background signal can be avoided by eliminatin the highly repetitive sequence component from the test sequence. See, for example, Sealey et al., Nuc. Acids Res., 13: 1905-1922 (1985).

Still another example is where the test sequence comprise a first subsequence which delivers only a small signal in a hybridization blot relative to the signal delivered by a second subsequence. Here, it may be more advantageous to eliminate the first test subsequence so that a more "cost-effective" probe which delivers a higher signal on a per nucleotide basis may be produced. A more •specific example of this type is where the first subsequence is a "single-copy" sequence, and the second subsequence is a Variable Tandem Repeat Sequence. On a per nucleotide basis, the second sequence delivers more signal whenever there are more than a single copy at a genetic locus.

Discrete polynucleotide subsequences may be obtained from a test sequence in a number of ways, and are well within the capability of an ordinarily skilled artisan. For example, one end of the test sequence may be progressively removed by an exonuclease or SI enzyme,

_* while the other end is being protected. Another example is digestion with a restriction enzyme. Other methods of obtaining subsequences are within the contemplation of the present invention.

After the less desirable subsequences have been eliminated from a test sequence, the remaining portion of the test sequence is used in familial tests for the determination of the nature of the^' genetic locus (loci) which it is capable of detecting as described above.

Finally, nearby genomic sequences (including nearby VTR sequences) may be reached by chromosome walk.

As discussed in the Background section of this application, duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and a certain degree of mismatch can be tolerated. Therefore, whenever a test sequence obtained as describe hereinabove has been determined to be useful in probin target polynucleotides of interest, mutations (both singl and multiple), deletions, insertions of the useful tes sequence, and combinations thereof, wherein sai mutations, insertions and deletions permit formation o stable hybrids with said target polynucleotide o interest, are part of the present invention. Mutations, insertions and deletions can be produced in a give polynucleotide sequence in many ways, and these methods ar known to an ordinarily skilled artisan. Other methods ma become known in the future. The known methods include, bu are not limited to: 1. determining analytically th sequence of a test sequence of the present invention, synthesize chemically or otherwise an artificial sequenc which is a mutation, insertion or deletion of the tes sequence; 2. using a test sequence of the present inventio to obtain via hybridization a genomic sequence or otherwis which is a mutation, insertion or deletion of the tes sequence; and 3. mutating, inserting or deleting a tes sequence in vitro or in vivo.

It is important to note that the mutational insertional, and deletional variants generated from given test sequence may be more or less efficient than th test sequences, in the sense that (a) more or fewe genetic loci may become detectable, (b) more or fewe alleles of a particular locus, may become detectable, (c more or less stable under stringent hybridizatio conditions, and (d) any combination of the above Notwithstanding such differences in efficiency, thes variants are within the scope of the present invention.

In another embodiment of the present invention, the useful test sequences described hereinabove are used fo genetic analysis, (i.e., used as probes), including bu not limited to analysis of genetic identity, relatedness or alteration. In one preferred embodiment, the method of genetic analysis comprises:

(a) digesting a DNA sample with a restriction endonuclease;

(b) separating the DNA restriction fragments according to size by electrophoresis;

(c) transferring the separated DNA in a state suitable for hybridization to a binding surface; (d) hybridizing the transferred DNA with a useful test sequence labeled with a signal-generating moiety, and

(e) detecting the signals generated; whereby the pattern of signals generated provides information about the composition of the DNA sample. This and other embodiments of the present invention involve the use of useful test sequences as probes in methods of genetic analysis.

EXAMPLES

Following are examples which illustrate procedures, including the best mode, for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 — Detection Of Genomic Sequences Which Hybridize With Oligomers Of A Consensus Sequence Of A VTR Human genomic DNA incompletely digested with the restriction endonuclease EcoRI was cloned into the bacteriophage lambda Charon 30. Restriction fragments ranging from 4.5 kbp to 17.5 kbp can be cloned into this vector. Gene 12: 301-309 (1980). About 5000 phage plaques were screened according to the method of Maniatis et al., supra, at page 321. Oligomers of 5'- CCCCCCGTGTCGCTGTT-3', the 17-base consensus sequence o VTR at the 3' end of the human alpha globin complex, wit an average length of 200-300 bases, were used to scree the phage "lifts".

Example 2 — Analysis Of Human Sequences In Recombinan Phaqes Whose DNA Hybridizes With Oligomers of VT Consensus Sequence A number of recombinant phages whose DNA hybridize with oligo-5'-CCCCCCGTGTCGCTGTT-3' were analyzed. Th results obtained with Phage 19 are presented below. DN from Phage 19 was extracted and digested with th restriction endonuclease EcoRI. The digests were subjecte to electrophoresis in an agarose gel. The electrophoreti pattern obtained is shown in Figure 1A. Lane M containe bacteriophage lambda HindiII fragments as molecular weigh markers. Bands A and B are the arms of the cloning phag vector. Bands C and D are human genomic sequence inserts. The DNA fragments in Figure 1A were transferred to nylon membrane, and probed with oligo-5 ' CCCCCCGTGTCGCTGTT-3 ' according to the procedure o Southern, supra. Figure IB shows the results of th hybridization blot. The numbers on the side of the Figur state the molecular weight in kilobase pairs. Only th larger human insert band, i.e.. Band C was detected. Se Figure IB. Therefore, only Band C contained human DN which is homologous with oligo-5'-CCCCCCGTGTCGCTGTT-3' .

Example 3 Human Genomic Sequence Inserts from Phage 1

As Polymorphic Probes

DNAs from Bands C and D of Figure 1A, representing the human genomic sequence insert in Phage 19, wer extracted separately. They were used separately in th Southern format to probe human genomic DNAs from 4 unrelated individuals.

The target human sequences were restriction fragments produced by digestion with one of the following restriction endonucleases: Pstl, Hinfl, Hindlll, EcoRI, Taql, Mspl, PvuII, Rsal, and BstNI.

The following experiment and results were representative. 5 ug of Pstl digested DNA from each of 4 individuals were electrophoresed and blotted onto a nylon membrane. Nylon membranes were prepared in duplicate. Either [³²P]-labeled DNA from Bands C of Figure 1A, or [^■ 2p]-labeled DNA from Band D of Figure 1A was mixed with excess unlabeled total human genomic DNA, and used to probe the target sequences on the membranes. Radioactive labeling was achieved by random 6-mer primed enzymatic synthesis, using radioactive precursors as substrates. However, other methods of labeling would also work as well. Total unlabeled human genomic DNA was added as a precautionary measure. It was known that the human genome contains widely dispersed highly repetitive sequences such as the Alu sequences. If the human insert in Phage 19 contained these and/or similar highly repetitive sequences, such repetitive sequences would produce a heavy background signal over the entire area on the blot where human target DNA could be found. The introduction of total human genomic DNA would serve to suppress this background signal. Sealey et al., supra.

The hybridization was carried out at 65^°C in 5X SSPE, 1-2% SDS (sodium dodecylsulfate), 0.5-1 mg/ml heparin. The blot was washed in O.IX SSC, 2.5 mM sodium phosphate, 1% SDS at 65^°C. [IX SSPE = 0.16 M NaCl, 0.01 M sodium phosphate, and 1 mM ethylenediaminetetraacetic acid. IX SSC = 0.15 M NaCl, 0.015 M sodium citrate.]

Figure 2 shows the results of these hybridization blots. Band C DNA was used in Figure 2A. Band D DNA was used in Figure 2B. In Figures 2A, the lane marked M contained lambda HindiII fragments as molecular weight markers, and the remaining four lanes contained Pstl digested DNA from unrelated individuals. The same preparation of human DNAs was used in Figure 2B.

An average of about 7-10 bands were detected in each individual when Band C DNA was used as probe. See Figure 2A. By contrast. Band D DNA could detect only a subset of the bands. See Figure 2B. The subset, indicated by the arrows, were present in all 4 unrelated individuals and would therefore appear to be non-polymorphic. The other bands were present in some but not all individual, and therefore, polymorphic. See Figure 2A. The fact that these polymorphic bands relate to multiple loci was confirmed in Example 10.

Band C human insert DNA was also able to detect multiple polymorphic loci when human genomic DNAs were digested with Hinfl, Hindlll, EcoRI, Taql, Mspl, PvuII, and Rsal. However, polymorphism was not detected when the human genomic DNA was digested with BstNI. Because geneti variations other than the VTR type are rare in man (about 0.001 per base pair), the probability that a non-VTR type polymorphism, i.e., a polymorphism caused by alterations i restriction sites in the neighborhood of the target sequence, would be revealed by at least seven restriction enzyme digestion is vanishingly small. Correlatively, the evidence is overwhelming that Band C DNA is detecting a VT type polymorphism. The BstNI data is consistent with the hypothesis that the VTR being detected here has an internal BstNI site. The results of Example 6 as well as partial sequencing data support this hypothesis.

Therefore, Band C DNA, but not Band D DNA is useful as a multi-locus, polymorphic probe. Example 4 — Cloning Of The Human "Polymorphic Probe" Sequence From Phage 19

DNA in Band C of Figure 1A, representing part of th human sequence insert in Phage 19 was extracted, and clone into Bluescript, which is a trademarked cloning vector o Stratagene, San Diego, CA 92121. Bluescript is derivative of M13 as well as of pBR322, and is about 3kbp long. The EcoRI insertion site was used. Several Bluescript recombinants containing Band C human DNA were obtained.

Two clones designated pAC329 and pAC344 were used to probe a panel of human genomic DNAs. DNA from 5 unrelated individuals were digested with Pstl and two duplicate filters were prepared for the Southern procedure. The hybridization conditions were the same as described in Example 3. Figures 3A and 3B show the results obtained when the inserts of pAC329 and pAC344 were used as probes respectively. It is readily seen that the same pattern (of multiple bands) were obtained. However, the pAC344 insert is slightly longer, and more sensitive, i.e., produces a stronger signal under comparable conditions.

A third Bluescript recombinant was able to detect only non-polymorphic bands, i.e. the banding patterns are identical in several unrelated individuals. (Data not shown).

Band C appears to have more than one species of DNA sequences each approximately 5 kbp long. This is consistent with the preferred cloning size of the Charon 30 vector (see Example 1 above). This Example shows that both pAC329 and pAC344 are useful as multi-locus, polymorphic probes.

Example 5 - Analysis Of Human DNAs In pAC329 And pAC344 pAC329 DNA was digested with EcoRI and the approximately 5 kbp human DNA insert was isolated. The same was done with pAC344 DNA. The purified inserts wer further separately digested with Rsal, and the products o the restriction digest was electrophoresed in a 1% agaros gel. The electrophoretic pattern is shown in Figure 4. Lane (M) contained molecular weight markers (lambda Hindll and phi-x Haelll fragments). Lane (a) containe restriction fragments from the insert of pAC329; and lan (b) contained those from the insert of pAC344.

DNA from each of the four marked bands shown i Figure 4 was isolated and used to probe unrelated, huma genomic DNAs in the Southern format. The hybridizatio conditions were as described in Example 3, except tha unlabelled human genomic DNA was not present in the prob mix: Relevant results of hybridization blots against Pst digested human genomic DNA are shown in Figure 5. Th general conclusion is that DNA from each of Bands 1, 2, and 4 are useful as multi-locus, polymorphic probes.

Figure 5A shows the hybridization results when DN from Band 1 of Figure 4 was used as the probe. Under th above-indicated hybridization conditions, if the prob contained highly repetitive sequences such as the "Al sequences", a background signal in addition to an specific bands might be produced, because the probe woul have hybridized to homologous highly repetitive sequence which are widely dispersed throughout the human genome Such background signal is indeed indicated in Figure 5A.

Figure 5B shows the hybridization results when DN from Band 2 of Figure 4 was used as the probe. Otherwise, the target and the hybridization conditions were identical In contrast with Figure 5A, background signals which ar indicative of the presence of highly repetitive sequence in the probe are not as evident here.

Similarly, Figures 5C and 5D show the respectiv results when DNA from Bands 3 and 4 were used as th respective probes. Figure 5D shows rather less backgroun signal than Figure 5C.

This Example shows that the subsequences of pAC329 an pAC344 represented by Bands 2 and 4 are improvements ove pAC329 and pAC344 as multi-locus, polymorphic probes.

DNA from Band 4,i.e., the second largest restrictio fragment from Rsal digestion of the human DNA insert i pAC344 was cloned into the Smal insertion site of

Bluescript by "blunt end cloning". The recombinant molecule has been designated pAC365.

Example 6 — Analysis Of pAC365

A restriction map of pAC365 DNA was obtained using a standard method. The results of the mapping is shown in Figure 6. The human sequence can be excised from the recombinant with EcoRI and BamHI in the form of a 1.35 kbp (approx.) fragment. The human sequence is flanked by 7 nucleotides on one end and 11 on the other both from the polylinker region of Bluescript. See Figure 6. The 1.35 kbp fragment was excised and used to probe

Pstl digested human genomic DNA identically prepared as in Example 5, and under identical conditions. The results of the hybridization is shown in Figure 7. A comparison of Figure 5D with Figure 7 shows that the signal patterns in the two blots were identical. This shows that the DNA sequence from Band 4 of Figure 4 was successfully cloned into pAC365.

The 1.35 kbp fragment was digested with BstNI and the restriction digest was electrophoresed in a 3% NuSieve agarose gel. [NuSieve is a trademarked product of FMC Corporation] . The electrophoretic pattern is shown in Figure 8. Lane M of Figure 8 contained phi-x Haelll molecular weight markers. The BstNI digest of the 1.35 kbp fragment shows a broad band at about 70-100 bp (indicated by an arrow in the Figure) and a band at a higher molecular weight of approximately 0.9 kbp. The sum of the molecular weights pertaining to these two bands is below 1.35 kbp. Therefore, the results indicate that the 1.35kbρ fragment comprises several copies of an approximately 70-100 bp VTR "core" sequence.

Example 7 — Analysis Of The Subfragments Of The pAC365 Insert

The 1.35 kbp human sequence cloned into pAC365 was excised from the plasmid along with 18 nucleotides from the polylinker region of Bluescript by digestion with EcoRI and BamHI. The excised fragment is further digested with one of the following enzymes: Pstl, Xbal, Sau3A I, Hinfl, and Ncol, thus generating five subfragments that each had the EcoRI site of 1.35kbp fragment at one end.

Each of these five subfragments were tested for its utility as multi-locus, polymorphic probes. Each is hybridized to a replicate panel of Pstl digested, unrelated, human genomic DNAs. The hybridization conditions were as described in Exam-pie 5. Parts of the results are shown in Figure 9. Figures 9A, and 9B show the results where EcoRI-Pstl, and EcoRI-NcoI subfragments respectively, were used as the probe. It is readily see that each of these subfragments generated the same pattern as the full pAC365 insert. Compare Figure 7 with Figures 9A, and 9B. Furthermore, the other subfragments also yielded the identical banding pattern. Hence, only th results for the largest (EcoRI-Pstl) and the smalles (EcoRI-Ncol) subfragments are shown. Finally, the DNA in the 100 bp area of Figure 8 was used as probe, and was able to generate an identical pattern on a replicate panel of target human DNA. This is further evidence that pAC365 contains a VTR sequence.

This Example shows that each of these subfragments ar useful multi-locus, polymorphic bands. Example 8 — Hybrid Formation And Stability Under High an Low Stringency Conditions

The 1.35 kbp insert of pAC365 was used to probe panel of Pstl digested human genomic DNA. The results ar shown in Figure 10. Figure 10A shows a Southern blo where hybrid formation took place at 50 C, in 5X SSPE, an the blot was washed at 65 C in 2X SSC. Figure 10B shows duplicate blot except it was washed at 65 C in O.IX SSC. No unlabeled total human genomic DNA was added to the probe mix.

It is believed that the probe contained a small amount of Alu-like sequence, and this accounts for the background signal. More significantly, the background is dramatically lower in Figure 10B than in Figure 10A. Most importantly, the specific signals, i.e., the polymorphic bands are strong and unambiguous when the blot was washed under high stringency conditions.

When hybrid formation and washing take place under more stringent conditions, background suppression is further improved. Figure 10C shows a blot where hybrids were formed at 65^°C in 5X SSPE, and where the blot was washed at 65°C in 0.1X SSC. The hybrids were actually stable at the even more stringent conditions: 0.01X SSC at 65^°C.

These results can be juxtaposed with those obtained using pAC256 as a probe. pAC256 is a Bluescript recombinant containing a human insert sequence which contains a Variable Tandem Repeat Sequence. See McClain et al., Am. J. of Human Genetics 41(Suppl. ) :A259 (1987). pAC256 behaves in conformity with prior art expectation. Prior art teaches that even though VTR probes can detect many loci at low stringency, the probes can only identify a single locus at high stringency. See, for example, Nakamura et al. (1987), Science 235: 1616, at 1618. Figure 11 illustrates just this point. Genomic huma DNA were extracted, digested with Pstl, and hybridized i the Southern format to pAC256 human insert at 65°C in 5 SSPE. Figure 11A shows the results when the blot was washe at 65°C in 2X SSC. Figure 11B shows the results when th blot was instead washed at 65°C in O.IX SSC. It is readil seen that many of the bands detected under the les stringent conditions were unstable under the more stringen conditions and were washed away. The fact that no mor than two bands were seen in each of the lanes containin target human DNA is consistent with the notion that at mos a single locus - the locus from which the probe sequenc originated - can be detected.

Example 9 — Equivalency Of Various DNA Sequences A Polymorphic Probes

The following polynucleotide sequences were tested fo differences, if any, in terms of the ability to detec polymorphism at multiple loci: DNA contained in Band C o Figure 1A ("Band C DNA"); 19-MSP (defined below); th insert of pAC365; and the 5 subfragments described i Example 7.

19-MSP was obtained from "Band C DNA" by digesting th latter with Mspl. "Band C DNA" appears to contain mor than one species of DNA sequences of about 5 kbp. On species is cut into several smaller pieces by Mspl. Thi species does not detect polymorphism. The other species i also cut by Mspl, but the larger digestion product is onl slightly smaller than 5 kbp. This approximately 5 kb product is designated 19-MSP.

19-MSP and "Band C DNA" detected the same polymorphi bands when tested against genomic DNA from more than twent genetically unrelated individuals. "Band C DNA" was abl to detect, in addition to these polymorphic bands, severa non-polymorphic bands. These results are illustrated i Figure 12. Figures 12A and 12B show Southern blots probe respectively with "Band C DNA" and 19-MSP, but are otherwise prepared in duplicate. Each blot contained Pstl digested genomic DNA from genetically unrelated individuals. When one of test lanes, for example, the rightmost test lane in Figure 12A is compared with its counterpart, the rightmost test lane of Figure 12B, it is seen that the former contain extra bands which are marked with arrows. However, these are non-polymorphic bands, as evidenced by the appearance of the same sized bands in all other test lanes in Figure 12A. These results show that "Band C DNA" and 19-MSP recognize the same DNA polymorphisms.

19-MSP and the insert of pAC365 detected the same polymorphic bands when tested against genomic DNA from 107 genetically unrelated individuals. These results show that the insert of pAC365 and 19-MSP are equivalent probes.

Similarly, each of the five subfragments described in

Example 7 have been shown to be equivalent to pAC365. (Representative data shown in Figures 7 and 9) .

Example 10 — pAC365 Detects Multiple Loci Which Segregate Independently In the Mendelian Fashion

Genomic DNAs were extracted from individuals belonging to families spanning three generations, digested with Pstl, and probed with pAC365 insert in the Southern Format. Figure 13 shows the results of one such family study. The family tree at the top of the figure indicate the source of DNA in each of the test lanes. The lanes marked (m) contained molecular weight markers.

A total of 27 bands of varying sizes were detected by hybridization with pAC365. These are partially numbered on the side of the figure for identification. Table 1 shows the phenotype of each individual. Several conclusions can be drawn from these results First, each and every band which is present in any one o the eight children is also present in either the father o the mother. This result is consistent with stabl chromosomal inheritance. Similarly, every band which i present in the father is present in either the paterna grandfather or grand-mother.

Second, the results are consistent with independen Mendelian segregation of alleles present on siste chromosomes. For example. Bands 1 and 4 appear to be tw alleles to the same gene. The mother has both of thes alleles, presumably one on each sister chromosome However, each of the eight children inherits one or th other allele, but never both. Thus, the mode o inheritance is consistent with independent Mendelia segregation. Similarly, Bands 10 and 17 in the fathe segregate in a manner which is consistent with the notio that they are two alleles on the same gene.

Table 1. InheritanceAnd Segregation Of Allele WithinA Family*

Family Member

1 2 3 4 5 6 7 8

Bands

1 + ₊ _ _ _ + _ _ __. + _ _ -

2 _ _ _ _ _ _ _ _ _ _ -. _ +

3 - - - + + + + - - + + - +

4 - + + + + - + + + - - - -

5 + _ _ _ _ _ _ _ _ _ ^' _ - _

6 - - - + - + + - - + + + -

7 _ + _ _ _ + _ + + _ _ + _

8 + _ _ _ _ _ _ _ _ _ _ _ - g . _ _{+ + + + +} _ _ _{+ +} _ _ _

10 - - - - + + + + - - + - +

11 + + - + + - - + - - + + -

12 __. _ _ _ _ _ _ _ _ _ _ + +

13 _ + + _ _ + _ _ _ + _ _ _

14 + + + + + + + + + + + - +

15 _ _ _ _ _ _ _ _ __. ' _ _ + _

16 _ _ _ __. _ _ _ _ _ _ __. _ +

17 -• - + + - - - - + + + + -

18 _ _ _ _ _ _ _ _ _ _ _ _ +

19 _ + + _ + + + _ _ + _ _ _

20 + _ _ _ _ _ _ _ _ _ _ + _

21 _ _ _ _ _ _ _ _ _ _ _ ₊ _

22 _ _ _ + ₊ _ _ _ _ _{+ +} _ ₊

23 + _ _ _ _ _ _ __. _ _ _ _ _

24 + + + + + + + + + + - - -

25 __. _ + _ _ + + + + _ ₊ + _^■'

26 + + + + + + + + + + + + +

27 + + + + + + + + + + + + +

* No data from maternal grandmother.

Third, the results are consistent with the notion that Band 1 is not linked to any of Band 7, 9, 13 or 19 These bands are all present in the mother. However, chil #8 inherited Band 1, but not Band 7 from the mother Therefore, Band 1 is not linked to Band 7. Child # inherited Bands 9, 13 and 19, but not Band 1 from th mother. Therefore, Band 1 is not linked to Band 9, 13 o 19.

Similar analyses lead to the conclusion that Band 7 i not linked to any of the other Bands present exclusively i the mother, nor Band 9, nor Band 13 and 19. Bands 1, 7, 9 13 and 19 represent five genetic loci.

Example 11 Population Genetics and Allele Frequency DNAs from 423 genetically unrelated individuals wer tested in this study. Each DNA sample was digested wit Pstl and probed in a Southern hybridization procedure. Fo 127 samples, the insert from. pAC365 was used as the probe For the other 296 samples, 19-MSP was used as the probe However, it had been established that the two probe recognize the same DNA polymorphisms. Therefore, poolin

_* of results obtained by using these probes is valid. Th pooled results have been sorted according to their ethni origins (i.e., American Blacks, Caucasoids, an Hispanics). The frequency v. allele size distribution are shown in Figure 14. Figures 14A, 14B, and 14C show th distributions in American Blacks, Caucasoids, and Hispanic respectively. The y-axis is measured .in per cent, and th x-axis is measured in kilobase pairs. The frequenc distributions are more fully set forth in Table 2. Table 2. Allele Frequencies For Three Racial Groups**

Fragment Size (kilobases)*** 2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65

3.50 3.55 3.60 3.65 3.70 3.75 3.80 3.85

4.80 4.85 4.90 4.95 5.00 5.05 5.10 5.15 5.20

5.25 5.30 5.35 5.40 5.45 5.50 5.55 5.60 5.65

5.70 5.75 5.80 5.85 5.90 5.95 6.0 6.1 6.2

6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1

7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9

Blacks 8 3 1 5 3 4 1 2 3

Caucasoids 1 2 1 2 2 1 1 2 2 Hispanics 1 5 2 1 1 0 0 4 1

9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8

Blacks 3 1 2 3 3 3 7 3 0

Caucasoids 2 1 2 4 1 2 1 1 2

Hispanics 1 2 2 1 1 0 0 0 1

9.9 10 10.2 10.4 10.6 10.8 11.0 11.2 11.4

Blacks 2 3 3 0 0 5 4 3 0

Caucasoids 1 2 7 7 2 5 7 2 1 Hispanics 2 2 6 2 2 4 6 5 3 11.6 11.8 12.0 12.2 12.4 12.6 12.8 13.0 13.2

Blacks 1 0 2 1 2 2 1 1 2 Caucasoids 2 1 2 1 1 1 2 1 1 Hispanics 3 0 0 1 2 0 0 0 0

13.4 13.6 13.8 14.0 14.2 14.4 14.6 14.8 15.0

15.2 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8

17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4

18.6 18.8 19.0 19.2 19.4 19.6 19.8 20.0

** Allele frequencies are stated in per cent in Table 2.

*** The standard error for fragment size is approximately 0.6% of the size of the fragment. Therefore, DNA fragments whose sizes are within 2% of each other (3 standard deviations) are considered indistinguishable. Example 12 Characterization of pAC365

Genomic DNA from various human cell lines were extracted, digested with Pstl, and hybridized with pAC365 insert in the Southern format. 7024, 7351, 7047, 7432, 7433 and 7015 were obtained from Centre d'Etude du Polymorphisme Humain in France. 1202 was obtained from the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository (Catalog Number 1202B). It is a lymphoblast cell line with 49 chromosomes (XXXXY). CEM and Jurket are T lymphoblastoid cell lines. K562 is a erythroleukemia cell line and HL60 is a promyelocytic cell line. CEM, K562, and HL60 can be obtained from the American Type Culture Collection ( "ATCC" ) under ATCC catalog numbers CCL119, CCL243, and CCL240, respectively. The bands detected in the Southern blot are set fort in Table 3 below. For example, pAC365 insert detected 9 bands ranging from 7.1 kilobase pairs to 2.2 kilobase pairs when hybridized with Pstl digested K562 cell DNA. The banding pattern obtained for each cell line is unique. Therefore, when used for probe purposes polynucleotide sequences can be characterized, or "fingerprinted" by th banding pattern with known target DNA.

Table 3. "Fin er rint" of AC365

# Sizes of the fragments detected are stated in kbp in Table 3.

Example 13 Paternity Testing and Forensic Testing

Genomic DNAs were extracted from a child, the mother of the child and the alleged father. The DNAs were digested with Pstl, electrophoresed and transferred for 5 Southern hybridization. The DNA targets were probed with 19-MSP. Figure 15A shows the results of the blot. Lanes labeled (m) contained molecular weight markers. Lanes (a), (b), and (c) contained DNA from the mother, the child, and the alleged father of the child respectively. Lane (d) 0 contained a mixture of the child's DNA and the alleged father's DNA. The last lane often helps to resolve ambiguity whenever a band detected in the child's lane is close in size to a band detected in the alleged father's lane. In such a case, the presence of a singlet band in 5 the relevant size region in the "child plus alleged father" lane would tend to indicate a common allele; and a doublet band would indicate distinct alleles.

Figure 15A shows that at least 5 bands in lane (b) were not inherited from the mother because they are not present in lane (a). However, each of these bands (marked with arrows) are present in lane (b). Therefore, the evidence supports the theory that the alleged father is indeed the biological father.

DNAs were extracted from a rape victim, semen found on the victim and from a suspect of the crime. The DNAs were digested with Pstl, and subjected to the Southern hybridization procedure. 19-MSP was used as a probe. Figure 15B shows the results of the hybridization blot. Lane (a) contained DNA from the victim. Lane (b) and (c) contained DNA from semen found on the victim, and from suspect, respectively. At least 8 bands (marked wit arrows) in lane (b) do not match the bands in lane (a), clearly indicating that these bands did not arise fro cells of the victim which some-how contaminated the seme sample. However, all 8 bands matched bands of the sam sizes in lane (c). Therefore, the evidence that the seme came from the suspect was exceedingly strong.

Example 14 Relationships Among The Polymorphic Probes O The Invention

Figure 16 shows the relationships among the various DNA sequences of the present invention, which are usefu as polymorphic probes.

DEPOSIT OF MICROORGANISM

Many polynucleotide sequences may be used to practic the present invention. Exemplary of such sequences ar human genomic sequences which have been cloned int recombinant plasmids designated pAC329, pAC344, and pAC365. Figure 16 shows the relationships among the clone sequences of this invention.

An E_. coli strain HB101 carrying the plasmid pAC329, an 13. coli strain HB101 carrying the plasmid pAC344, and an E . coli strain HB101 carrying the pAC365 plasmid have been deposited with the Agricultural Research Culture

Collection (NRRL), Peoria, IL, on September 2, 1988, and have been assigned accession numbers NRRL B-18403, NRRL B-

18404, and NRRL B-18405, respectively. The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and

Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed.

However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent dislcosing them.

E. coli HB101 is available from the NRRL repository where its accession number is NRRL B-11371. Plasmids can be isolated from the E___ coli host by use of standard procedures, e.g., using cleared lysate-isopycnic density gradient procedures, and the like.

The present invention is not to be limited in scope by the microorganisms deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention. Many variations of this invention as herein set forth may be made without departing from the spirit and scope thereof. The specific embodiments described are given by way of example only, and the invention is limited only by the terms of the appended claims.

Claims

Claims

1 1. A polynucleotide sequence capable of forming

2 hybrids with genomic Variable Tandem Repeat DNA sequences

3 at multiple, polymorphic genetic loci of a eukaryote under

4 hybridization conditions wherein said hybrids are stable

5 under high stringency conditions.

1 2. The polynucleotide sequence of claim 1 wherein the

2 eukaryote is a mammal.

1 3. The polynucleotide sequence of claim 2 wherein the

2 mammal is a human.

1 4. A polynucleotide sequence represented by the human

2 DNA insert in pAC329.

1 5. A polynucleotide sequence represented by the human

2 DNA insert in pAC344.

1 6. A polynucleotide sequence represented by the

2 restriction fragment of about 1.35 kilobase pairs produced

3 by digestion of pAC365 with EcoRI and BamHI.

1 7. A polynucleotide sequence represented by the 2 restriction fragment of about 0.55 kilobase pairs produced 3 b y digestion of pAC365 with EcoRI and Ncol.

1 8. A discrete polynucleotide subsequence of the

2 polynucleotide sequence of any of the claims 4 to 7,

3 wherein said subsequence is capable of forming hybrids with

4 genomic DNA at one or more polymorphic genetic loci of a

5 eukaryote, and mutational, insertional or deletional

6 variants thereof.

9. The discrete polynucleotide subsequence of claim 8 wherein the eukaryote is a human.

10. A polynucleotide sequence capable of forming a hybrid with the polynucleotide sequence of any of th claims 4 to 7, wherein the hybrid is stable under hig stringency conditions.

11. A polynucleotide sequence capable of forming hybrid with the discrete polynucleotide subsequence o with its variants of claim 8, wherein the hybrid is stabl under high stringency conditions.

12. A polynucleotide sequence capable of formin hybrids with genomic DNA fragments, produced by complet digestion of Caucasoids* , American Blacks' and Hispanics' genomic DNAs with the restriction endonuclease Pstl, o approximate lengths and approximate allele frequencies a described in Table 2.

13. A polynucleotide sequence useful for detectin polymorphism at multiple genetic loci, and characterized b its ability to form hybrids with restriction fragments o DNA, of 7.1, 6.2, 4.4, 4.2, 4.1, 3.7, 3.6, 2.6 and 2. kilobases, produced by Pstl digestion of genomic DN extracted from K562 cells.

14. A recombinant vector comprising the polynucleotid sequence of any of the claims 4 to 7.

15. A recombinant vector comprising the polynucleotid subsequence of claim 8.

16. A recombinant vector comprising the polynucleotid sequence of claim 10.

17. A recombinant vector comprising the polynucleotide sequence of claim 11.

18. A recombinant vector comprising the polynucleotide sequence of claim 12 or 13.

19. A cell containing the recombinant vector of claim 14.

20. A cell containing the recombinant vector of claim 15.

21. A cell containing the recombinant vector of claim 16.

22. A cell containing the recombinant vector of claim 17.

23. A cell containing the recombinant vector of claim 18.

24. A method for obtaining the polynucleotide sequence of claim 1 comprising: (a) hybridizing a library of genomic sequences of an organism under hybridization conditions with a probe comprising a natural or consensus sequence of a Variable Tandem Repeat; (b) selecting library sequences of said library which hybridize with said probe; (c) testing and further selecting from the library sequences selected in step (b) a library sequence which is capable of detecting Variable Tandem Repeat Length polymorphism at multiple genetic loci of said organism or a different organism under high stringency conditions.

25. A method for obtaining the polynucleotide sequence of claim 1 comprising: (a) hybridizing a library of genomic sequences of an organism under hybridization conditions with a probe comprising a natural or consensus sequence of a Variable Tandem Repeat; (b) selecting library sequences of said library which hybridize with said probe; (c) producing subsequences of said selected library sequences by enzymatic means; (d) testing and selecting from the subsequences produced in step (c) a subsequence which is capable of detecting Variable Tandem Repeat Length polymorphism at multiple genetic loci of said organism or a different organism under high stringency conditions.

26. A polynucleotide probe comprising the polynucleotide sequence of any of the claims 4 to 7, or mutational, insertional, or deletional variants thereof.

27. A polynucleotide probe comprising the discrete polynucleotide subsequence of claim 8, or mutational, insertional, or deletional variants thereof. '

28. A polynucleotide probe comprising the discrete polynucleotide subsequence of claim 10, or mutational, insertional, or deletional variants thereof.

29. A polynucleotide probe comprising the polynucleotide sequence of claim 11, or mutational, insertional, or deletional variants thereof.

30. A polynucleotide probe comprising the polynucleotide sequence of claim 12 or 13, or mutational, insertional, or deletional variants thereof.

31. A method of genetic analysis comprising: (a) digesting a DNA sample with a restriction endonuclease; (b) separating the DNA restriction fragments according to size by electrophoresis; (c) transferring the separated DNA to a binding surface; (d) hybridizing the transferred DNA with a polynucleotide probe labeled with a signal- generating moiety, wherein the polynucleotide probe is the polynucleotide probe of claim 26; and (e) detecting the signals generated; whereby the pattern of signals generated provides information about the composition of the DNA sample.

32. A method of genetic analysis comprising: (a) digesting a DNA sample with a restriction endonuclease; (b) separating the DNA restriction fragments according to size by electrophoresis; (c) transferring the separated DNA to a binding surface; (d) hybridizing the transferred DNA with a polynucleotide probe labeled with a signal- generating moiety, wherein the polynucleotide probe is the polynucleotide probe of claim 27; and (e) detecting the signals generated; whereby the pattern of signals generated provides informatio about the composition of the DNA sample.

33. A method of genetic analysis comprising: (a) digesting a DNA sample with a restrictio endonuclease; (b) separating the DNA restriction fragment according to size by electrophoresis; (c) transferring the separated DNA to a bindin surface; (d) hybridizing the transferred DNA with polynucleotide probe labeled with a signal generating moiety, wherein the polynucleotid probe is the polynucleotide probe of claim 28 and (e) detecting the signals generated; whereby th pattern of signals generated provides informatio about the composition of the DNA sample.

34. A method of genetic analysis comprising: ( a) digesting a DNA sample with a restrictio endonuclease; (b ) separating the DNA restriction f ragment according to size by electrophoresis; ( c ) transferring the separated DNA to a bindin surface; (d) hybridizing the trans f erred DNA with polynucleotide probe labeled with a . signal generating moiety, wherein the polynucleotid probe is the polynucleotide probe of claim 29 and (e) detecting the signals generated; whereby th pattern of signals generated provides informatio about the composition of the DNA sample. A method of genetic analysis comprising: digesting a DNA sample with a restriction endonuclease ; separating the DNA restriction f ragments according to size by electrophoresis; transferring the separated DNA to a binding surface; hybridi z ing the trans ferred DNA wi th a polynucleotide probe labeled with a signal- generating moiety, wherein the polynucleotide probe is the polynucleotide probe of claim 30 ;

detecting the signals generated; whereby the pattern of signals generated provides information

about the composition of the DNA sample.