WO1995027077A1

WO1995027077A1 - Y chromosome minisatellite

Info

Publication number: WO1995027077A1
Application number: PCT/GB1995/000706
Authority: WO
Inventors: Mark Alexander Jobling; Nourdine Bouzekri
Original assignee: University Of Leicester
Priority date: 1994-03-31
Filing date: 1995-03-29
Publication date: 1995-10-12
Also published as: AU2078095A; GB9406534D0

Abstract

The present invention provides a Y chromosome minisatellite comprising a 5' flanking sequence, a repeat unit or tandem repeat units, and a 3' flanking sequence. Also provided are repeat unit variants and amplification primers for the minisatellite and repeat unit variants, together with MVR-PCR primers for same.

Description


  
 



   Y Chromosome Minisatellite
 This invention concerns isolated polymorphic sequences on the Y chromosome and DNA probes thereto, particularly though by no means exclusively, for use in forensic male-specific identification or exclusion, or in genealogical applications in societies where surname and Y chromosome are coinherited.



   The study of paternal lineages and population histories through the analysis of modem Y chromosomes has been hampered by the lack of useful polymorphisms on the Y chromosome. Known RFLPs (Restriction Fragment Length Polymorphisms) are either dimorphic, or complex and difficult to interpret. Several long-range multiallelic polymorphisms have been described but these are technically difficult to type, and there is no practicable method available for the study of mutation processes at these loci.



   Ideal polymorphisms would (i) have many alleles; (ii) be amenable to PCR typing ; (iii) permit direct observation of mutation rates and processes in sperm; (iv) have alleles which are very unlikely to occur independently in separate lineages.



   Minisatellites on the Y chromosome would be expected to fulfill the criteria for such polymorphisms but have hitherto not been described.



   The present invention identifies a minisatellite on the Y chromosome thus providing sites on the Y chromosome having polymorphisms for use in individual identification and paternal lineage studies.  



   Minisatellites are loci consisting of tandem arrays of short (10-50bp) repeat units, found in the genomes of most higher eukaryotes. The number of these units is commonly variable, and the consequent array length polymorphism has been exploited in linkage analysis (Donis-Keller etal., 1987, Cell 51: 319-337), individual identification by DNA fingerprinting (Jeffreys et al., 1985, Nature 316 : 76-79), and in population genetic studies (eg. Flint et al., 1989, Hum. Genet. 83: 257-263).



   Minisatellites tend to be clustered at the ends of human chromosomes and are common in the short arm of pseudoautosomal region of the sex chromosomes.



  However, this is the first time that such a minisatellite has been described in the
Y-specific DNA.



   As well as their value in studying paternal lineages, such sequences would be forensically useful in male-specific identification and in genealogical studies in societies where surname and Y chromosome are coinherited.



   Additionally the minisatellite sequence of the present invention may be used to investigate mutation processes since variation in the sequence of individual repeat units within the minisatellite exists, and this can be conveniently accessed by the technique of minisatellite variant repeat PCR (MVR-PCR; Jeffreys et al., 1991, Nature 354: 204-209), in which PCR primers designed to discriminate between different repeat types allow the generation of products extending to a fixed flanking primer, thus mapping the positions of particular variant repeats within the array (see Figure 1). This allows for a digital typing of a region of DNA and extremely easy comparison of digital codes between individuals.  



   MVR-PCR reveals the enormously high degree of variability at these loci and has allowed the details of mutation processes to be investigated. Mutations to new length arrays occur predominantly in the germ line, with a frequency of about 1%, and a preponderance of repeat gains over losses. All minisatellites investigated so far show a strong polarity in germline mutation, with a high frequency of recombination and gene conversion events occurring at one end of the array, which has led to the suggestion that these inter-allelic exchanges are driven by a cis-acting element in the flanking DNA (Jef & eys et al., 1994, Nature Genet. 6: 136-145).



   According to the present invention there is provided an isolated
Y-chromosome minisatellite comprising:
 i) a 5'flanking sequence comprising the DNA sequence of SEQ ID NO: 1;
 ii) a repeat unit, or tandem repeat units; and
 iii) a 3'flanking sequence comprising the DNA sequence of SEQ ID NO: 2 and homologues thereof.



   The repeat unit or tandem repeat units may be 25 bp in length. The minisatellites so far isolated and sequenced have contained repeat units all of which are 25 bp in length, indicating that length polymorphisms of the repeat units may be infrequent.



   There may be between 1 and 100 repeat units in the minisatellite.



   The repeat unit or units may be selected from the group of SEQ ID NO: 3,  
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID No: 13.



   Initial work resulted in the isolation of five variation repeats (SEQ ID No's 3,4,5,6 and 7; also referred to as Type 1-5 variants respectively) and further work has resulted in the isolation of six further repeat variants (SEQ ID No's: 8,9,10,11,12 and 13; also referred to as Type 6,7,8,9, A and B variants respectively). Additional variants exist and they may be readily isolated using the methods detailed below.



   The variants may be specific to particular families and population lineages.



   These variants may be used to type Y chromosomes from different males.



  Such a typing system may be used in forensic individual identification or genealogical studies.



   Also provided are amplification primers specific to the minisatellite of the present invention. The primers may be PCR primers. As such, they may be used as DNA probes for the minisatellite or for particular variants.



   A PCR primer may be specific to the 5'flanking sequence of SEQ ID
NO: 1; it may comprise the sequence of SEQ ID NO: 14 (also referred to as primer Y1A).



   A PCR primer may be specific to the 3'flanking sequence of SEQ ID
NO: 2; it may comprise the sequence of SEQ ID NO: 15 (also referred to as primer Y1B).  



   A primer may be specific to the repeat unit of any one of the group of SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID No: 13.



   It may be specific to the repeat unit of SEQ ID NO: 3 and comprise the sequence of SEQ ID NO: 16 (also referred to as TAG1).



   It may be specific to the repeat unit of SEQ ID NO: 5 and comprise the sequence of SEQ ID NO: 17 (also referred to as TAG3).



   It may be specific to the repeat unit of SEQ ID NO: 6 and comprise the sequence of SEQ ID NO: 18 (also referred to as TAG4).



   It may be specific to the repeat unit of SEQ ID NO: 3; and comprise the sequence of SEQ ID NO: 19 (also referred to as TAG1R).



   It may be specific to the repeat unit of SEQ ID NO: 5; and comprise the sequence of SEQ ID NO: 20 (also referred to as TAG3R).



   It may be specific to the repeat unit of SEQ ID NO: 6; and comprise the sequence of SEQ ID NO: 21 (also referred to as TAG4R).



   These primers (TAG1,3,4,1R, 3R and 4R) comprise a 5'20bp TAG sequence (see Experimental Data) and a sequence specific to the respective repeat unit variants.  



   A PCR primer may be specific to a repeat variant with a length other than 25 bp.



   Such a PCR primer may be used in a method of MVR-PCR.



   Also provided are pairs of amplification primers specific to the minisatellite of the present invention. The amplification primers may be PCR primers.



   Such pairs of PCR primers may be used in a method of MVR-PCR.



   Such a pair of MVR-PCR primers may comprise a primer specific to the flanking sequence of either one of SEQ ID NO: 2 and SEQ ID NO: 3 and a discriminator primer specific to a repeat unit; the repeat unit may be selected from any one of the group of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ
ID N0: 13.



   Such a pair of MVR-PCR primers may for example comprise a primer of
SEQ ID NO: 14 and a discriminator primer selected from any one of the group of SEQ
ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 or they may for example comprise a primer of SEQ ID NO: 15 and a discriminator primer selected from any one of the group of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.



   Apair of MVR-PCR primers may comprise a discriminator primer specific  to a repeat variant with a length other than 25 bp.



   A pair of PCR primers according to the present invention may comprise a pair of primers specific to the 5'flanking sequence of SEQ ID NO: 1 and the 3'flanking sequence of SEQ ID NO: 2.



   Such a pair of PCR primers may comprise a primer of SEQ ID NO: 14 and a primer of SEQ ID NO: 15.



   Other primers specific to any one of SEQ ID NO's 1-21 for use according to the present invention may be easily generated.



   A minisatellite, primer or pairs or primers according to the present invention may be used in a method of treatment or diagnosis of the human or animal body.



   Also provided according to the present invention is a method of treatment of the human or animal body comprising the use of a minisatellite, primer or pair of primers according to the present invention.



   The invention will be further apparent from the following description of experimental data and figures wherein,
 Figure 1 shows the principle of minisatellite variant repeat coding by
MVR-PCR;  
 Figure 2 shows an autoradiograph showing size variability of the 50f2/B EcoRI fragment in 19 unrelated Y chromosomes, and schematic positions of the 5 loci on the Y;
 Figure 3a shows 640bp partial sequence of 890bp HaeIII/HindIII fragment from the MSY1 locus. The MSY1 repeats, 14bp repeats and poly (A) tract, and positions of primers A and B are marked. Only three 25bp repeats are shown: the two most 5'are Type 1 and the most 3'is Type 2;
 Figure 3b shows predicted hairpin structures for Type 1 and 2 MSY1 repeats;
 Figure 4 shows EtBr stained gel of male-specific PCR products obtained with MSY1 flanking primers Y1A and Y1B. Normal males give two products, a 196bp and a variable product.

   The 196bp product is absent from male 38 and from TAP (46, XY, del (Y) (ql 1.1)), who lack 50f2/C, while the larger fragment is absent from 445/393, a hybrid lacking short arm material. Products are also obtained from cosmids containing the 50f2/B (heterogenous in size due to rearrangement), C (expected size of 196bp) and, weakly, D (about 1.3kb), and E (about 1.0kb) loci.



  Cosmids were isolated from the library LLOYNC03;
 Figure 5 shows a schematic representation of the MSY1 locus, showing organisation within the cosmid M29Gl 1, the five repeat types defined by an initial study, and the positions of primers used in the MVR-PCR assay (not to scale). The  type 5 repeat is unique to a single individual and is not typed. Type 2 repeats have been found in only two individuals so far and are not typed.



   Figure 6 shows repeat variant types at MSY1. The figure shows all repeat types found to date and indicates their distribution in the males studied.



   Figure 7a shows three-state MVR-PCR codes at MSY1. Codes for 27 alleles are shown, belonging to Y chromosome haplotypic groups 1 and 2. Of the populations, Eng=English; Ind=Indian subcontinent; Mal=Malaysian; Jap=Japanese;
Chi=Chinese; IJ=Iraqi-Jewish; Fin=Finnish; Rus=Russian; GleGreek; nk-not known. Y chromosomal haplotype Groups are as defined by Jobling (1994, Hum. Molec. Genet.



  3: 107-114)
 Figure 7b shows 16 further three-state MVR-PCR codes. The 16 alleles belong to Y chromosome haplotypic groups 3,4 and no defined group. Of the populations, Eng=English; Ind=Indian subcontinent; Ken=Kenyan; Nig=Nigerian;
Bia=Biaka Kung; Mal=Malaysian; Aus=Australian aboriginal; nk=not known.



   Figure 8 shows the sequences and positions of discriminator primers used in MVR-PCR at MSY1. Three repeat units of each repeat type (1,3 and 4) are shown in capitals (one strand only). Forward primers (lower case) are shown above the repeats and reverse primers below. Note: the reverse discriminators anneal to the other
 strand of the repeat array (sequence not shown). The 5'TAG sequence is that of Jeffreys
 et al., 1991, Nature, 354 : 204-209.  



   EXPERIMENTAL DATA
 The Y chromosome minisatellite sequence of the present invention (MSY1;
D-segment number DYFlSSSI allocated by Genome DataBase) was isolated and characterised using a probe 50f2 (DYS7; Guellaen et al, 1984, Nature 314: 67-73). 50f2 detects five Y-specific EcoRI fragments (Figure 2) corresponding to five distinct loci
A-E. The size of the second largest fragment, 50f2/B, was observed to vary in length between 7.5 and 8.5kb in a set of nineteen chromosomes (Figure 2), suggesting to the present inventors that a minisatellite of limited allele length diversity might lie within this EcoRI fragment.



   To isolate this putative minisatellite, the arrayed Y-specific cosmid library
LLOYNC03 was screened with 50f2, and a cosmid (M29G11) containing the B locus isolated. An unstable Hindi fragment of 3.8kb was subcloned, and 640bp of an 890bp
HaeMMindIH sub-fragment sequenced. This sequence (Figure 3a) is novel and contains a number of tandem repetitive elements, including a 23 residue poly (A) tract, a tandem pair of perfect 14bp repeats and an adjacent array of 25bp repeats which may vary in number from for example 1 to 100 due to variation in copy number of individual alleles.



   Two sequence variants, differing by a single transition, can be seen in the cloned array, which has lost approximately 60 repeats with respect to the source chromosome (chromosome 50 of Mathias et al., 1984, Hum. Mol. Genet. 3: 115-123), in hybrid J640-51). These repeats are A+T rich and have the potential to form cruciform structures (Figure 3b).  



  PCR Amplification of MSYI
 PCR primers (Y1A and Y1B) were designed flanking the array (Figure 3a) and used to amplify the locus from genomic and cosmid DNA (Figure 4).



   PCR across MSY1 gave poor product yield, probably due to the A+T rich and palindromic nature of the repeat.



   To identify repeat variants as a suitable basis for MVR-PCR, individual alleles were amplified with primers Y1A and Y1B and analysed without cloning using
Taq cycle sequencing and 33P end-labelled primers. This procedure typically gave 150-250bp of sequence from the 3'end, and 500-600bp from the 5'end. Variant repeats could be provisionally typed further into the array simply by reading the G-and Ctracks, where complete sequence was not readable. All repeats read in this sequencing were 25bp in length and the regular periodicity of the sequencing ladders suggests that no variant length units exist in the analysed arrays.



   In order to identify repeat variants in the minisatellite which could be used for digital coding with MVR-PCR, an initial study was performed in which individual alleles were amplified with primers Y1A and Y1B and analysed without cloning using
Taq cycle sequencing and 33P end-labelled primers. This procedure typically gave 150250 bp of sequence from the 3'end and 500-600bp from the 5'end. Variant repeats were provisionally typed further into the array by simply reading the G-and C-tracks where complete sequence was not readable.  



   This initial study led to the identification of five variant repeats (Types 1-5;
Figures 5 and 6). Three-state MVR-PCR coding of alleles revealed a number of'nulls'repeats which failed to code, or which coded anomalously faintly. In order to understand the sequence basis of these nulls, alleles were amplified and sequenced directly from their edges. This analysis has so far revealed an additional six repeat types (Types 6,7,8,9, A and B; Figure 6). Further routine analysis should reveal other repeat variants.



   Of the six additional repeats, three were originally identified as being nulls by MVR-PCR whilst three (9, A and B) were found to code as types 1,3 and 4 respectively but share an additional substitution (T-C) at position 6 of the repeat unit.



  These types appear to be confined to African Group 4 chromosomes and may be used in a test for such chromosomes.



   Additionally,'nulls'may have particular value in identifying specific sublineages.



   Studies performed so far (Figures 7 and 8) have used forward and reverse three-state MVR-PCR coding for types 1,3 and 4 (Figure 8) and have provided codes which cover most alleles examined in their entirety (58-96 repeats), giving a full picture of code diversity. Results for forty-seven three state codes show that these repeat types appear to be the commonest in most populations studied. Of the codes so far obtained, all are different, but many fall into groups of related structures which generally reflect groups of Y chromosomes defined by compound haplotyping by other means (Oakey,
R, and Tyler-Smith, C., 1990, Genomics 7: 325-330; Jobling, M. A., 1994, Hum. Molec.



  Genet., 3: 107-114; Mathias, N., et al., 1994, Hum. Molec. Genet., 3: 115-123). For  example, the twelve Group 1 (Caucasian-specific) chromosomes have closely related codes of 72-78 repeats, with the general tripartite structure (1) 15-17 (3) 3742 (4) lus23, while the five Group 3 (mostly Indian sub-continental) chromosomes have larger (93-96 repeats) tripartite alleles with the general structure (1)21-22(3)55-58(4)15-17. Group 2 (ethnically diverse) and Group 4 (mostly African) chromosomes have been found to be more heterogeneous, consistent with known haplotype data.



  MVR-PCR at MSY1
 MVR-PCR atMSYI is potentially problematic-discriminator primers will be A+T-rich, and therefore have relatively low annealing temperatures; because of the palindromic nature of the repeat unit, primers will contain regions of self-complementarity, and may dimerise, or form hairpin structures; and primers designed to discriminate between the variant repeats have to rely on internal mismatches for discrimination.



   An MVR-PCR system was designed to map forward into the 5'end and backward into the 3'end of the MSY 1 array. The system uses primers (Y1A and Y1B) specific to either one of the flanking sequences together with discriminator primers (TAG1,3,4,1R, 3R and 4R) specific to the repeat variants. The discriminator primers comprise the 20bp TAG primer sequence of Jef & eys et al (1991, Nature, 354 : 204-209) 5'of a discriminator sequence (Figure 8) in order to maximise annealing temperature and thus specificity.  



   The MVR-PCRprimers specific for Types 1,3 and 4 repeats discriminated well and yielded three-state MVR maps. These maps are consistent with sequencing data. Such maps may be used to type individual Y chromosomes for use in forensic applications in male-specific identification, particularly in mixed assailant-victim samples in rape cases, and in genealogical studies in societies where Y chromosome and surname are coinherited.



   MATERIALS AND METHODS
Standard procedures
 Genomic Southern analysis was carried out as described (Chen et al., 1992,
Nature Genet, 1,204-208)); cosmid library screening and subcloning into pBluescript II (RTM; Stratagene) were by standard techniques (Sambrook et al., 1989, Molecular
Cloning: a laboratory manual, 2nd Edn, Cold Spring Harbour Laboratory Press, NY);
DNA sequencing of single-and double-stranded subclones was by the dideoxy method (Sanger et al., 1977, PNAS 74: 5463-5467).



  PCR Amplification across MSY1
 All PCR reactions used the buffer system described by Jeffreys et al. (1990,
Cell, 60: 473-485), and were carried out in a Perkin-Elmer-Cetus 4800 thermocycler.



   Primers used were Y1A and Y1B (SEQ ID NO's 14 and 15 respectively).  



   Conditions for amplification from 100ng of genome template in 10111 reactions were as follows:
 Primer YlA-Y1B: 95 C 1 minute, 66"C 3.5 minutes, 25 cycles
Tag cycle sequencing ofalleles
 MSY1 alleles were amplified using primers A and B in 10 ul PCR reactions. MSY1 product was purified following agarose gel electrophoresis, reamplified, and repurified. 200ng of each allele were then reamplified asymmetrically, using either
A or B in a 30gel, 20-cycle PCR, to yield single-stranded product. This was recovered by isopropanol precipitation to remove remaining primers, and resuspended in 8111 water.



  1p1 ofthis was used in each of four 7.5 p1 Taq cycle sequencing termination PCRs, each containing a different dideoxy nucleotide. These reactions contained 33P-end-labelled primer (opposite to that used in the asymmetric amplification) at a concentration of 66nM, 45mM Tris-Cl (pH 8.8), 1 lmM ammonium sulphate, 7 mM 2-mercaptoethanol, 4.4 pM EDTA, 113 llg/ml BSA, 8pm each of dATP, dCTP, dTTP and dGTP and one of the ddNTPs at the following concentrations: 80lit ddGTP, 25011M ddATP, 330liM ddTTP, or 160uM ddCTP. After 10 cycles the PCR products were denatured and immediately loaded onto a 6% denaturing polyacrylamide gel.



  MVR-PCR at MSY1
PCR primer pairs were as follows:  
Forward coding: Y1A (SEQ ID NO: 14) used in conjunction with discriminator primers
TAG1,3 and 4 (SEQ ID NO's 16,17 and 18 respectively).



  Reverse coding: Y1B (SEQ ID NO: 15) used in conjunction with discriminator primers
TAG1R, 3R and 4R (SEQ ID NO's 19,20 and 21 respectively).



  MSY1 MVR-PCR was performed as follows:
 100ng genomic DNA were used as template in I Ogl PCR reactions using either Y1A or Y1B at a concentration of 100nM, plus a complementary discriminator primer at 100nM and 0.5 unit Taq polymerase. Amplification was carried out by an initial phase: denaturing at 95  C for 1 minute, annealing at 62  C for 1 minute and extending at 68 C for 3.5 minutes, repeated for 3 cycles; followed by a second phase: denaturing at 95  C for 1 minute, annealing/extending at 68OC for 4.5 minutes, repeated for 27 cycles.



   During initial studies, PCR products were electrophoresed for 14 hours at 2.5V/cm through a 35cm long 1.3% agarose (HGT; FMC Biologicals) gel in 89mM tris-borate (pH 8.3), 2mM EDTA (TBE). The gel was transferred by Southern blotting for 4-6 hours in 0.4M NaOH to Hybond N+ membrane (RTM; Amersham) and the filter neutralised in 0.2M Tris-Cl (pH 7.5), 2 x SSC before hybridisation. Hybridisation was in church buffer (Church  &  Gilbert, 1994, PNAS 81: 1991-1995) at 65 C overnight using 32P-multiprime labelled probe (see below) and washing to 0.2 x SSC at the same temperature. Autoradiography was at-80 C with an intensifying screen for 2-24 hours.



   The probe used was an approximate 1.3kb RsaI fragment including about  22 MSY1 repeats: this probe contains the material between the nearest 3'-flanking RsaI site and the 5'-flanking HindIII site. The RsaI site at the other end of the probe derives from the polylinker of the vector (pBluescript II; RTM)
 During further investigations (see Figures 7a and 7b), improved quality of coding was obtained by end-labelling the complementary discriminator primers with 33P.



  PCR products were denatured and immediately loaded onto a 2.5% denaturing polyacrylamide gel which was then dried down and exposed to X-ray film for 24-72 hours. This improved quality also offers the possibility of automation of MSY1 coding using dye-labelled primers and an automated sequence analyser.



   It will be appreciated that it is not intended to limit the invention to the above examples only, many variations, such as might readily occur to one skilled in the art being possible, without departing from the scope thereof.  



   SEQUENCE LISTING (1) GENERAL INFORMATION:
 (i)APPLICANT:
 (A) NAME: The University of Leicester
 (B) STREET: University Road
 (C) CITY: Leicester
 (E) COUNTRY: GB
 (F) POSTAL CODE (ZIP): LEI 7RH
 (A) NAME: JOBLING, Mark Alexander
 (B) STREET: 31 Thurlow Road
 (C) CITY: Clarendon Park
 (D) STATE: Leicester
 (E) COUNTRY: GB
 (F) POSTAL CODE (ZIP): LE2 1YE
 (A) NAME: BOUZEKRI, Nourdine
 (B) STREET: 4, Place du General Duval, No. 23
 (C) CITY: Aigues-Mortes
 (E) COUNTRY: France
 (F) POSTAL CODE (ZIP): 30220
 (ii) TITLE OF INVENTION: Y Chromosome Minisatellite
 (iii) NUMBER OF SEQUENCES: 21
 (iv) COMPUTER READABLE FORM:
 (A) MEDIUM TYPE: Floppy disk
 (B) COMPUTER: IBM PC compatible
 (C) OPERATING SYSTEM: PC-DOS/MS-DOS
 (D) SOFTWARE: PatentIn Release   #1.0,   Version   #1.30   (EPO)
 (vi) PRIOR APPLICATION DATA:
 (A) APPLICATION NUMBER:

   GB 9406534.9
 (B) FILING DATE: 31-MAR-1994 (2) INFORMATION FOR SEQ ID NO: 1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 393 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAGCTTTGGT TTGTAGGAAT TAGTGTCAAC.

   ATTCTAATAA TTATTTGGTA ATAACCCTGT 60
AGTTGGTTGA ATTCTCTAAA AAAAAAAAAA AAAAAAAAAA CTATGTTGAA ATAACCCTGA 120  
GGACCTGTGA CTATAACCTT ATTTGGAAAT ATAAACTTTT CAAATATAAT CAAGTTAAGG 180
TAAGTCACAT TACATTATGG TATACCCTAA ATTCAATGTC TTATATTGTT ATAAGGATAG 240
AGAGTTTTAG AGACACAGAG ACACAGAGGG AAGACAGCCA TGTGAAGACA GAGGTAGATG 300
CTGAAGCGGT ATAGCTACAA GCTGATGAAT GCCAAGGATA TATTATGCCA ATACATATAT 360
GCCAATACAT ATACTATGTA TATGTATAAT ATA 393 (2) INFORMATION FOR SEQ ID NO: 2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 172 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 2:
CATATGCACA CATAAACCCC TTTGAATAAA TAGTATTACG CTTCCCTTTC CCTTTGTCCT 60
AGCTTGAGTT GCCCAGAAAA CAAGTCTGAG GTTTGTTTAG TGGGGTATAT GCGATACAGG 120
ACAAGCAACA CATGGAAGGA AGGAGATTTA CTATAACATT TTATCAAGTA GG 172 (2) INFORMATION FOR SEQ ID NO: 3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CACAATATAC ATGATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 4:  
CATAATATAC ATGATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CACAATATAC ATCATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CATAATATAC ATCATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (gendmic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 7:
CATAATATAC ATGATGTATA ATATA 25 (2) INFORMATION FOR SEQ ID NO: 8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)  
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CACAATATAC TTGATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CACAATATAC ATCATGTATA ATATA 25 (2) INFORMATION FOR SEQ ID NO: 10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 10:
CACAACATAC ATCATGTATA ATATA 25 (2) INFORMATION FOR SEQ ID NO: 11:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CACAACATAC ATCATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 12:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double  
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CACAACATAC ATGATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 13:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 25 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 13:
CATAACATAC ATCATGTATA TTATA 25 (2) INFORMATION FOR SEQ ID NO: 14:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 24 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GAGGTAGATG CTGAAGCGGT ATAG 24 (2) INFORMATION FOR SEQ ID NO: 15:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 24 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTCAAGCTAG GACAAAGGGA AAGG 24 (2) INFORMATION FOR SEQ ID NO: 16:  
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 47 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: 

   SEQ ID NO: 16:
TCATGCGTCC ATGGTCCGGA TGTGTATAAT ATACATCATG TATATTG 47 (2) INFORMATION FOR SEQ ID NO: 17:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 47 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TCATGCGTCC ATGGTCCGGA TGTGTATAAT ATACATGATG TATATTG 47 (2) INFORMATION FOR SEQ ID NO: 18:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO: 18:
TCATGCGTCC ATGGTCCGGA CATGATGTAT ATTATGTATA ATATACATG 49 (2) INFOR 
TCATGCGTCC ATGGTCCGGA CACAATATAC ATGATGTATA TTATACAC 48 (2) INFORMATION FOR SEQ ID NO: 20:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
TCATGCGTCC ATGGTCCGGA CATCATGTAT ATTATACACA ATATACATC 49 (2) INFORMATION FOR SEQ ID NO: 21:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: DNA (genomic)
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TCATGCGTCC ATGGTCCGGA CATCATGTAT ATTATACATA ATATACATC 49

Claims

CLAIMS 1. An isolated Y-chromosome minisatellite comprising: i) a 5'flanking sequence comprising the DNA sequence of SEQ ID NO: 1; ii) a repeat unit, or tandem repeat units; and iii) a 3'flanking sequence comprising the DNA sequence of SEQ ID NO: 2 and homologues thereof.

2. A minisatellite according to Claim 1 wherein the repeat unit or tandem repeat units are 25bp in length.

3. A minisatellite according to either one of Claims 1 or 2 wherein the number of repeat units is between 1 and 100.

4. A minisatellite according to any one of Claims 1-4 wherein the repeat unit or units are selected from the group of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

5. An amplification primer specific to the minisatellite of any one of Claims 1-4.

6. An amplification primer according to Claim 5 wherein it is a PCR primer.

7. A PCR primer according to Claim 6 wherein it is specific to the 5' flanking sequence of SEQ ID NO: 1.

8. A PCR primer according to Claim 7 wherein it comprises the sequence of SEQ ID NO: 14.

9. A PCR primer according to Claim 6 wherein it is specific to the 3' flanking sequence of SEQ ID NO: 2.

10. A PCR primer according to Claim 9 wherein it comprises the sequence of SEQ ID NO: 15.

11. A PCR primer according to Claim 6 wherein it is specific to the repeat unit selected from any one of the group of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

12. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 3 and comprises the sequence of SEQ ID NO: 16.

13. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 5 and comprises the sequence of SEQ ID NO: 17.

14. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 6 and comprises the sequence of SEQ ID NO: 18.

15. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 3 and comprises the sequence of SEQ ID NO: 19.

16. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 5 and comprises the sequence of SEQ ID NO: 20.

17. A PCR primer according to Claim 11 wherein it is specific to the repeat unit of SEQ ID NO: 6 and comprises the sequence of SEQ ID NO: 21.

18. A PCR primer according to Claim 6 wherein it is specific to a repeat variant with a length other than 25 bp.

19. A PCR primer according to any one of Claims 7 to 18 wherein it is used in a method of MVR-PCR.

20. A pair of amplification primers specific to the minisatellite of any one of Claims 1-4.

21. A pair of amplification primers according to Claim 20 wherein they are PCR primers.

22. A pair of PCR primers according to Claim 21 wherein they are used in a method of MVR-PCR.

23. A pair of MVR-PCR primers according to Claim 22 comprising a primer specific to the flanking sequence of either one of SEQ ID NO: 2 and SEQ ID NO: 3 and a discriminator primer specific to a repeat unit.

24. A pair of MVR-PCR primers according to Claim 23 wherein the repeat unit is selected from any one of the group of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

25. A pair of MVR-PCR primers according to Claim 24 comprising a primer of SEQ ID NO: 14 and a discriminator primer selected from any one of the group of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

26. A pair of MVR-PCR primers according to Claim 24 comprising a primer of SEQ ID NO: 15 and a discriminator primer selected from any one of the group of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

27. A pair of MVR-PCR primers according to Claim 23 wherein the discriminator primer is specific to a repeat variant with a length other than 25 bp.

28. A pair of PCR primers according to Claim 21 wherein they are specific to the 5'flanking sequence of-SEQ ID NO: 1 and the 3'flanking sequence of SEQ ID NO: 2.

29. A pair of PCR primers according to Claim 28 wherein they comprise a primer of SEQ ID NO: 14 and a primer of SEQ ID NO: 15.

30. A minisatellite, primer or pair of primers according to any one of preceding claims when used in a method of treatment or diagnosis of the human or animal body.

31. A method of treatment of the human or animal body comprising the use of a minisatellite, primer or pair of primers according to any one of Claims 1-29.