The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.

There is considerable interest in understanding patterns of linkage disequilibrium (LD) in the human genome, to aid investigations of human evolution and facilitate association studies in complex disease. The relative influences of meiotic crossover distribution and population history on LD remain unclear, however. In particular, it is uncertain to what extent crossovers are clustered into 'hot spots, that might influence LD patterns. As a first step to investigating the relationship between LD and recombination, we have analyzed a 216-kb segment of the class II region of the major histocompatibility complex (MHC) already characterized for familial crossovers. High-resolution LD analysis shows the existence of extended domains of strong association interrupted by patchwork areas of LD breakdown. Sperm typing shows that these areas correspond precisely to meiotic crossover hot spots. All six hot spots defined share a remarkably similar symmetrical morphology but vary considerably in intensity, and are not obviously associated with any primary DNA sequence determinants of hot-spot activity. These hot spots occur in clusters and together account for almost all crossovers in this region of the MHC. These data show that, within the MHC at least, crossovers are far from randomly distributed at the molecular level and that recombination hot spots can profoundly affect LD patterns.

Simple tandem-repetitive regions of DNA (or 'minisatellites') which are dispersed in the human genome frequently show substantial length polymorphism arising from unequal exchanges which alter the number of short tandem repeats in a minisatellite. We have shown previously that the repeat elements in a subset of human minisatellites share a common 10-15-base-pair (bp) 'core' sequence which might act as a recombination signal in the generation of these hypervariable regions. A hybridization probe consisting of the core repeated in tandem can detect many highly polymorphic minisatellites simultaneously to provide a set of genetic markers of general use in human linkage analysis. We now show that other variant (core)n probes can detect additional sets of hypervariable minisatellites to produce somatically stable DNA 'fingerprints' which are completely specific to an individual (or to his or her identical twin) and can be applied directly to problems of human identification, including parenthood testing.

PRDM9 has recently been identified as a likely trans-regulator of meiotic recombination hot spots in humans and mice1-3. The protein contains a zinc finger array that in humans can recognise a short sequence motif associated with hot spots4, with binding to this motif possibly triggering hot-spot activity via chromatin remodelling5. We now show that variation in the zinc finger array in humans has a profound effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Very subtle changes within the array can create hot-spot non-activating and enhancing alleles, and even trigger the appearance of a new hot spot. PRDM9 thus appears to be the preeminent global regulator of hot spots in humans. Variation at this locus also influences aspects of genome instability, specifically a megabase-scale rearrangement underlying two genomic disorders6 as well as minisatellite instability7, implicating PRDM9 as a risk factor for some pathological genome rearrangements.

Many highly polymorphic minisatellite loci can be detected simultaneously in the human genome by hybridization to probes consisting of tandem repeats of the 'core' sequence. The resulting DNA fingerprints produced by Southern blot hybridization are comprised of multiple hypervariable DNA fragments, show somatic and germline stability and are completely specific to an individual. We now show that this technique can be used for forensic purposes; DNA of high relative molecular mass (Mr) can be isolated from 4-yr-old bloodstains and semen stains made on cotton cloth and digested to produce DNA fingerprints suitable for individual identification. Further, sperm nuclei can be separated from vaginal cellular debris, obtained from semen-contaminated vaginal swabs, enabling positive identification of the male donor/suspect. It is envisaged that DNA fingerprinting will revolutionize forensic biology particularly with regard to the identification of rape suspects.

Meiotic gene conversion has an important role in allele diversification and in the homogenization of gene and other repeat DNA sequence families 1-5 , sometimes with pathological consequences 6,7 . But little is known about the dynamics of gene conversion in humans and its relationship to meiotic crossover. We therefore developed screening and selection methods to characterize sperm conversions in two meiotic crossover hot spots in the major histocompatibility complex (MHC) 8 and one in the sex chromosomal pseudoautosomal pairing region PAR1 (ref. 9). All three hot spots are active in gene conversion and crossover. Conversion tracts are short and define a steep bidirectional gradient centered at the peak of crossover activity, consistent with crossovers and conversions being produced by the same recombination-initiating events. These initiations seem to be spread over a narrow zone, rather than occurring at a single site, and seem preferentially to yield conversions rather than crossovers. Crossover breakpoints are more broadly diffused than conversion breakpoints, suggesting either differences between conversion and crossover processing after initiation or the existence of a quality control checkpoint at which short interactions between homologous chromosomes are preferentially aborted as conversions.Meiotic gene conversion in humans is defined as the recombinational transfer of information between alleles or loci without crossover. (This definition, though widely used, is not necessarily congruent with conversion classically defined by non-2:2 meiotic segregation ratios and does not include crossovers accompanied by conversion.) Homogenization events in multigene and dispersed repeat sequence families and in palindromic DNA 3-5 , as well as the direct detection of de novo conversion events in families 6,10 and in sperm 11,12 , provide extensive evidence for interlocus conversion. Indirect evidence for interallelic conversion comes from seemingly anomalous low levels of linkage disequilibrium seen between some closely linked markers 13 and from patchwork patterns of allelic diversity 14,15 (though such patchworks could arise from sequential crossovers). Sperm analysis at the HLA-DPB1 locus has provided direct evidence that true interallelic conversions do occur, though at low frequency 16 . The relationship between crossover and conversion in humans, however, is unclear.The high-resolution definition of human crossover hot spots by sperm typing 8,9,15 has allowed us to investigate the connection between crossover and interallelic conversion without crossover. We chose the hot spot DNA3 located in the MHC for analysis 8 , because of its intense crossover activity and the availability of sperm donors with multiple single nucleotide polymorphism (SNP) heterozygosities needed to monitor recombination events. Our initial approach was similar to that used to detect conversion in HLA-DPB1 (ref. 16; Fig. 1a). We amplified pools of sperm DNA by PCR using allele-specific primers outside the hot spot to amplify one haplo...

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Tandem-repetitive minisatellite regions in vertebrate DNA frequently show substantial allelic variation in the number of repeat units. This variation is thought to arise through processes such as unequal crossover or replication slippage. We show here that the spontaneous mutation rate to new length alleles at extremely variable human minisatellites is sufficiently high to be directly measurable in human pedigrees. The mutation rate at different loci increases with variability in accord with the neutral mutation/random drift hypothesis, and rises to 5% per gamete for the most unstable human minisatellite isolated. Mutations are sporadic, occur with similar frequencies in sperm and oocytes, and can involve the gain or loss of substantial numbers of repeat units, consistent with length changes arising primarily by unequal exchange at meiosis. Germline instability must therefore be taken into account when using hypervariable loci as genetic markers, particularly in pedigree analysis and parenthood testing.