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Biology and applications of human minisatellite loci
Biology and applications of human minisatellite loci John A.L. Armour and Alec J. leffreys U n i v e r s i t y of Leicester, Leicester, UK
Highly repetitive minisatellites' include the most variable human loci described to date. They have proved invaluable in a wide variety of genetic analyses, and despite some controversies surrounding their practical implementation, have been extensively adopted in civil and forensic casework. Molecular analysis of internal allelic structure has provided detailed insights into the repeat-unit turnover mechanisms operating in germline mutations, which are ultimately responsible for the extreme variability seen at these loci.
Current Opinion in Genetics and Development 1992, 2:850-856
Introduction Polymorphism in DNA structure provides the basis of genetic analysis. The first human DNA-sequence variants to be analyzed directly were base substitutional polymorphisms affecting the recognition sites for restriction enzymes [1]. As these restriction-site dimorphisms can only have two allelic states, their informativeness is limited to a ma.ximum heterozygosity of 50%. Following the first description of a highly polymorphic locus in human DNA [2], further examples of loci at which multi-allelic variability in allele length (Fig. 1) was caused by differences in the numbers of short repeated units were described
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[3-5]. Tandem repetition is widespread in the human and other eukaryotic genomes with variation in the number of tandemly repeated units occurring on many different scales. At one extreme, the alphoid satellites of human centromeres can have arrays up to 5 Mb in total length [6], and pulsed-field gel electrophoresis analysis has shown variation in the array length at different centromeres [7,8]. At the other end of the scale, arrays of dinucleotide repeats [9,10] and even mononucleotide repeats in retroposon tails [ 11 ] frequently show variation in the number of repeat units. Here, we will discuss the biology and applications of hypervariable 'minisatellites', at which total array size is generally in the range 0.5-30kb. The discovery and use of probes that are able to simultaneously detect large numbers of hypervariable tandemly repeated minisatellite loci have shown that they are widespread in the human genome [12], and have provided the basis for the systematic isolation of minisatellites by cloning [13-16,17,18oo].
Fig. 1. Schematic representation of the general principle of allele length variation caused by variation in tandem repeat copy number. Three different alleles are shown, containing five, eight or nine repeat units (black arrows). The consequent variation in the length of the array can be assayed as restriction fragment length variation by using Southern-blot hybridization after digestion with a restriction enzyme, such as X used here, that does not cut within the repeat array. Alternatively, relatively short arrays can be amplified using flanking single-copy primers, such as A and B (open arrows).
Minisatellites in genetic analysis Multi-locus DNA fingerprinting Using tandemly repeated probes in hybridization at low stringency, it has been demonstrated that highly variable tandemly repetitive loci are abundant in the hu-
Abbreviation MVR--minisatellite variant repeat.
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Biology and applications of human minisatellite loci Armour, Jeffreys 851 man genome [12]. The simultaneous detection of many hypervariable loci has also provided a powerful tool in genetic analysis. Because the combinatorial resolution of large numbers of highly polymorphic loci make the profile individual-specific, the profile is called the 'DNA fingerprint' or 'genetic fingerprint' [19]. In addition to these probes (ultimately derived from an intron in the human myoglobin gene; see [12]), other naturally occurring [20,21] and synthetic [22,23] tandem repeats have been successfully used to detect multiple hypervariable loci in human DNA. Individual-specific, multi-locus DNA fingerprints have a wide variety of applications, particularly in the establishment of family relationships [24,25 °] and to a lesser extent in forensic identification [26]. The high germline mutation rate at hypervariable minisatellite loci causes occasional anomalies in parentage analysis [12], but new mutations can almost always be distinguished with confidence from incorrect paternity ([25.]; see below). The obvious exception to individual specificity is in the case of monozygotic twins; indeed, the determination of zygosity in twins is an important special application of DNA fingerprinting [27]. In addition to their uses in human genetic analysis, multi-locus DNA fingerprints also have important applications in non-human species [28--31].
PCR typing at tandemly repeated loci An additional level of sensitivity can be achieved by using DNA amplification. PCR analysis of length variation at highly informative minisateUite loci is possible, but suffers from the disadvantage that many alleles at highly polymorphic loci are too large (e.g. 10-20kb) to amplify efficiently [40]. A few minisatellite loci combine high informativeness with restricted allele size, such that complete PCR profiles can be obtained [41%42]. The more abundant dinucleotide [9,10] and simple tandem repeat [43"*] loci can be typed by PCR to give phenotypes that, in principle, can be defined precisely by allele size; the small size of alleles (typically 100-200 bp) also means that they will be relatively resistant to DNA degradation in forensic specimens, as demonstrated by two extreme cases [44"',45"]. However, the informativeness of microsatellite loci is relatively limited, such that multiple loci often need to be typed to give results of significant statistical weight [44"]; furthermore, the systematic generation of series of artefactual 'slippage' products during the amplification of short repeat arrays [9,10] can introduce uncertainty in evidentiary use. PCR-based typing of highly variable minisatellites by sampling internal repeatunit variants (minisatellite variant repeat (lVlVR)-PCR; see below) has the advantages of sensitivity, extreme variability and digital encoding of phenotype, and can be applied to the analysis of partially degraded DNA [46.*].
Single locus profiles Hypervariable minisatellite loci studied singly, using cloned locus-specific probes in hybridization at high stringency, have a wide variety of applications as highly informative genetic markers. In segregation analysis, for example, analysis of linkage to a genetic disease is made more efficient by using extremely informative loci (for example, see [32]). Their use in general linkage analysis is however limited by their uneven distribution in the genome: while minisatellites appear to be approximately equally represented on all human autosomes, most hypervariable loci studied by in situ hybridization have been localized to subtelomeric regions [18",33,34]. Similarly, hypervariable loci mapped by linkage preferentially localize to near the ends of linkage maps [17,18",35,36]. Although similar clusters seem to exist in cattle [31], minisatellites do not appear to localize to subtelomeric regions in mice [37]. The high level of informativeness at many minisateUites nevertheless makes them very useful in a wide variety of genetic analyses. Their efficiency in distinguishing between the two alleles of an individual at a specific locus is of particular importance in detecting allele losses from tumours [38] and in the detection of karyotypic abnormalities such as uniparental disomy [39"]. In the deterruination of individual identity from forensic specimens, the use of single loci has the advantage that results can be obtained from less DNA than required for multi-locus DNA fingerprinting, and from DNA mixtures in forensic casework [14]; the DNA profiles obtained are also simpler to interpret, and can be databased as estimated allele lengths.
Controversies in evidentiary use Laboratory practice and data interpretation The use of minisateUite loci as evidence in legal proceedings, usually in the determination of paternity or individual identification in forensic medicine, has generated considerable debate on the reliability of DNA profiling in general and the use of minisatellite loci in particular [47]. While not placing undue emphasis on "exaggerated concerns over minor imperfections" [48], the potential problems in the evidentiary uses of minisatellite loci deserve discussion in a review of their applications. Many of the material criticisms of DNA profiling have centred on the results of relaxed laboratow practice, and of low or inconsistent standards of data interpretation - - primarily matters for standards of accreditation and quality control in the laboratories involved. Of particular concern has been the need to establish an objective set of criteria sufficient to declare a match between a forensic specimen and a suspect's DNA. The problem is that bands on an autoradiograph may appear to match, but can strictly only be said to be indistinguishable at the resolution afforded by the gel system used. Indeed, there are circumstances, for example with a badly contaminated forensic specimen, in which alleles may migrate to different distances on the gel, despite coming from the same person ('bandshift'; [49"]). Furthermore, the degree of precision with which the apparent match can be defined determines in part the statistical weight attached to the result; the tighter the 'window' within which bands from the two matching samples must lie, the more unlikely it is that the match is fortuitous.
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Genomesand evolution At some loci, a relatively small number of discretely identifiable alleles differing by integral numbers of repeat units may, in principle, be assayed with precision [41,]; however, the existence of even uncommon repeat units of non-standard sizes or base composition can generate anomalous 'non-integral' alleles. The usual compromise made at loci with essentially continuous allele size distributions, such as most minisatellites used in DNA profiling, is to make the conservative declaration that the two samples match to within a certain distance, say 1 mm, on the gel. This assumption of imprecision can then be built into the statistical appraisal of the result (for example, see [25 °] ). An extension of this approach is the use of predefined size intervals or 'bins' into which alleles are classified ([50"]; but see also [51]).
Mutation and population substructuring In addition to the issues of good laboratory practice and primary data interpretation raised above, the use of minisatellite profiling as evidence may be susceptible to two important sources of possible (non-human) error: gross variation in allele frequencies between subpopulations, and, in paternity disputes, germline mutation [52]. There is a theoretical possibility that mosaicism for a somatic mutation may lead to discrepancies in individual identification, but this would act to produce a false exclusion rather than a spurious inclusion [53]. The potential influence of population substmcturing derives from an assumption that the tendency of germline mutation to diversify minisatellite alleles is significantly counteracted by the tendency of genetic drift to fix alleles within a subpopulation. Thus, population substructure might lead to a particular DNA profile being less rare within, for example, an ethnic minority or genetically isolated population than in the larger population from which the allele-frequency estimates were derived. Empirical analysis of multi-locus DNA fingerprints, based on extensive paternity casework, suggests that paternity can be established with confidence even in the presence of germline mutations [25"]; furthermore, at least within the (Caucasian) populations studied, population substructuring does not appear to exert significant influence on the statistical weight of results. Although a number of studies have directly addressed this issue (for example, [54] ), the empirical evidence is much less clear for minisatellite loci examined singly, and the extent to which population heterogeneity influences the statistical confidence of DNA profiling results has been a matter of dispute [55-.,56.o,57.]. The extremely (some would say excessively) conservative approach suggested by Lewontin and Hard [55"'] includes the need for databases appropriate to each ethnic group, and the empirical estimation of probabilities that make no assumptions about the population genetics of minisatellite loci. For example, a phenotype not hitherto encountered in a database of N individuals would have its frequency conservatively estimated as 1/N [55"']. These population genetic issues, as well as specific recommendations for conservative approaches to the estimation of genotype frequencies from
allele frequencies, have recently been discussed in a National Academy report on DNA technology in forensic science [58°]. The practical difficulty of such a conservative approach, namely the need to assemble large databases, based on error-prone allele-length measurement, for each locus and each ethnic group, may in principle be mitigated by the adoption of digital phenotyping systems such as MVRPCR [46..]. A rapid typing system is used in MVR-PCR to assay a second level of variability within minisatellite arrays, the distribution of different types of repeat unit along an allele (see below and Fig. 2). Genomic DNA is typed directly, to give a diploid profile resulting from the superimposition of the two allelic patterns. These extraordinarily variable, digitally encoded phenotypes have the advantage that the data are unambiguous, providing simple match criteria, and are 'portable' - - there is no need for 'side by side' comparison to establish identity or exclusion, and data can be exchanged between centres, allowing the cumulative assembly of large phenotypic databases.
Length-change mutation and the origin of variation General parameters of the mutation process Allelic diversity at minisatellite loci is generated by a high rate of spontaneous germline mutation to new-length alleles. At extremely variable loci, germline mutations to alleles containing new numbers of repeat units occur at a rate high enough to measure by direct observation in pedigree analysis [59]. The observed mutation rates to new-length alleles correlate with heterozygosity as predicted by a model of neutral mutation and random drift. While at most loci mutations appear to arise with similar frequency in male and female germlines, at least one locus shows a predominance of mutations originating in the male germline [18,.]. Although germline mutation is the ultimate source of allelic variability in populations, somatic mutations can also occur at minisatellite loci [53,60]. Somatic instability is a prominent feature of two mouse minisatellites [61,62.], at which somatic mutation early in development gives rise to mice globally mosaic for mutant and non-mutant cells. Somatic deletion mutants at the minisatellite D1S8 (MS32) in humans have also been recovered from blood DNA by using size-selection followed by single molecule dilution and PCR [63°']. The apparent relative stability in the population of small minisatellite alleles provides indirect evidence that the germline mutation process may operate at a frequency dependent on allele length [64.]. Similarly, the threshold effects seen at trinucleotide arrays associated with the fragile-X syndrome and myotonic dystrophy [65"] also suggest a strong correlation between allele length and mutation rate for these disease loci. Analysis of cognate minisatellite loci in non-human primates likewise suggests that short arrays can be stable over millions of years [66°].
Biology and applications of human minisatellite loci Armour, Jeffreys
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Fig. 2. Minisatellite variant repeat (MVR) mapping. Many minisatellite loci have alleles composed of an interspersed mixture of two or more different types of repeat unit. In this example, the two repeat-unit types are distinguished by shading. The pattern of interspersion of the repeat-unit types (the 'internal map' or 'MVR map') can in some cases be assayed using restriction mapping. (a) Enzyme E2 has sites (indicated by vertical bars) in both types of repeat; however, only the shaded variant repeat contains a site for enzyme El. Thus, mapping sites for El can be used to map the positions of a particular repeat-unit type. (b) A more efficient method, MVR-PCR, uses PCR primers to discriminate between repeat-unit types. MVR-map variation represents a second level of variation within minisatellite alleles: for example, the two alleles shown below have the same number of repeat units (19), but their internal maps show that they are clearly distinct alleles. Thus, an individual with these two alleles would appear to be a homozygote for allele length, but is in fact heterozygous, as shown by MVR mapping. Digital DNA typing uses MVR-PCR to analyze both alleles simultaneously to generate the digital code indicated: I, both repeats unshaded; 2, both shaded; and 3, heterozygous.
The mechanisms by which minisatellite-length changes may occur include replication slippage, intramolecular recombination, unequal sister-chromatid exchange, and unequal inter-allelic recombination or gene conversion. The approximately equal frequency of mutations increasing or decreasing allele length excludes intramolecular recombination as the predominant mechanism [59]. Analysis of a single germline mutation at D17S5 (YNZ22) showed that polymorphisms flanking the minisatellite were not exchanged during the mutation process, thus excluding a simple recombinational model for that event [67]. Similarly, analysis of a series of mutations at DIS7 (MS1) failed to show any increase in recombination between distal flanking markers associated with mutation
[68].
Internal m a p p i n g of minisatellite
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More detailed analysis of minisatellite variation and mutation has been possible by studying a second level of variation at minisatellite loci. In addition to allelic variation in the number of tandem repeats, all minisatellites that have been extensively studied show subtle variations in the precise sequence of repeat units [12-14,69-71], such that a minisatellite allele is frequently composed of an interspersed mixture of two or more MVRs (Fig. 2) [46°.,63°.]. The patterns of interspersion of variant repeats were initially ascertained using restriction-site mapping [63o°], but MVR-PCR has recently provided a much more rapid method for MVR mapping [46o°].
Studies of the internal disposition of variant repeats within alleles shows that there is variation in internal map structure as well as in length; indeed, internal map variation can be truly enormous, with perhaps as many as 108 different alleles at D1S8 in the present world population [46°°]. More surprisingly, there is a gradient of variability; at one end of the locus, the pattern of repeat units shows only a small number of distinct types in different alleles, while the other end is 'ultravariable', with nearly each allele having its own unique pattern. While this polarity was detected first in the MS32 (D1S8) minisatellite [46°,,63°°], there is evidence for similar gradients of variability at two other loci (JAL Armour, DL Neff and AJ Jeffreys, unpublished data). This preferential localization of variability at one end of minisatellite loci predicts that germline mutations would be localized at. this end of the array, and analysis of germline mutations at D1S8 and other minisateUite loci shows that germline mutation events are indeed concentrated in the first few repeats at the 'ultmvariable' end [46 °°] (JAL Armour and AJ Jeffreys, unpublished data). This region, then, appears to be a hot-spot for length-change mutations, and suggests that the mutation process at these loci may be under the influence of some as yet unidentified c/s-acting element. By comparing the maps of new mutant and progenitor alleles, internal mapping of minisateUite structure also allows conjectures to be made about the mechanisms of minisateUite mutation. This more recent work shows that; while the majority of mutation events appear to be entirely intra-allelic, some events can only be satisfactorily explained as the result of inter-aUelic exchanges between
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Genomes and evolution
parental alleles. This observation may parallel other evidence implicating minisatellites in recombinational processes and in genomic instability [33,72.o,73-75], and suggests further that the nautational hot-spot at one end of these arrays may also be a hot-spot for meiotic recombination and/or gene conversion [46-. ] (JAL Armour and AJ Jeffreys, unpublished work).
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NAKAMURAY, LEPPERT M, O'CONNELL P, WOLFF R, HOhM T, CUL\q?.R M, MARTINC, FUJIMOTO E, HUFF M, KUMIJN E, WHITE R: Variable Number of Tandem Repeat (VNTR) Markers for Human Gene Mapping. Science 1987, 235:1616-1622.
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Conclusion
The extension of minisatellite profiling to MVR analysis promises a wealth of detailed information on their applications in genetic analysis, including evidentiary uses, as well as in the elucidation of mutational mechanisms.
Acknowledgements We thank D Monckton for helpful comments on time manuscript. JAL Armour is a Wellcome Trust postdoctoral fellow. Work carried out in AJ Jeffreys group is supported by grants from tile MRC and the Royal Society.
VERGNAUDG, MARIAT D, APIOU F, AURIAS A, LATHROP M, LAUTHIERV: The Use of Synthetic Tandem Repeats to Isolate New VNTR Loci: Cloning of a Human Hypermutable Sequence. Genomics 1991, 11:135-144. Describes the isolation of hypervariable loci from human DNA by hybridization with syntheUc tandem repeats. One of the loci isolated is extremely unstable; mutation occurs preferenUally in timemale gemmline, with a mutation rate to new-length alleles of 15% per spemm. 18. ida
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JEFFREYSAJ, WIL¢~ONV, NEUMANNR, KEY'rEJ: Amplification of H u m a n Minisatellites by the Polymerase Chain Reaction: Towards DNA Fingerprinting of Single Cells. Nucleic Acids Res 1988, 16:10953-10971.
41.
BUDOWLEB, CHAKRABORTYR, GIUSTI AM, EISENBERG AJ, ALLEN RC: Analysis of the VNTR Locus D1SSO by the PCR Followed by High-Resolution PAGE. Am J H u m Genet 1991, 48:137-144. Describes the PCR typing of a highly informative locus to give discretely classifiable alleles. .
42.
BOERWINKLEE, XIONG W, FOOREST E, CHAN L: Rapid Typing of Tandemly Repeated Hypervariable Loci by the Polymerase Chain Reaction: Application to the Apolipoprotein B 3' Hypervariable Region. P m c Nail Ac ad Sci USA 1989, 86:212-216.
EDWARDSA, CmTELLO A, HAMMONDHA, CASKEYCT: DNA Typing and Genetic Mapping with Trimeric and Tetrameric T a n d e m Repeats Am J H u m Genet 1991, 49:746-756. Demonstrates the utility of arrays composed of tri- or tetranucleotide repeats as informative multiallelic genetic markers.
43. °.
44. .°
HAGELBERGE, GRAY IC, JEFFREYS AJ: Identification of the Skeletal Remains of a Murder Victim by DNA Analysis. Nature 1991, 352:427-429.
55.
LEWOr, mN RC, HARTL DL: Population Genetics in Forensic DNA Typing. Science 1991, 254:1745-1750. his work details the population genetic basis for reservations about some current practices in the forensic use of minisatellite typing. 56. CHAKRABORTYR, KJDD KK: T h e Utility of DNA typing in .• Forensic Work. Science 1991, 254:1735-1739. An answer to some of the points raised by Lewontin and Hard [55°°]. The (controversial) circumstances surrounding the publication of this article are discussed in the News a n d Comment section of this same issue of Science. Correspondence arising from this, and from Lewontin and Hard's article, appear in the 28 February 1992 issue of Science. See
also [57"]. 57.
BROOKFmLD J: Law and Probabilities. Nature 1992, 355:207-208. Summarizes the Lewontin and Harfl [55 °°] vs Chakrabony and Kidd [56 *° ] debate regarding DNA typing in forensic law, and describes the surrounding controversy. ,
58, w
NATIONALRESEARCHCOUNCIL,COMMITTEEON DNA TECHNOLOGY IN FORENSIC SCIENCE (McKuSlCK VA, CHAIRMAN):DNA Technol-
855
856
Genomes and evolution ogy in Forensic Science. Washington DC: National Academy Press; 1992. A comprehensive review of many aspects of the use of DNA typing in forensic studies. The report endorses the general use of DNA typing in forensic science, and makes specific recommendations for conservative approaches to the statistical evaluation of typing results.
59.
60.
61.
JEFFREYS AJ, ROYLE NJ, WILSON V, WONG Z: Spontaneous Mutation Rates to New Length Alleles at Tandem-repetitive Hypervariable Loci in H u m a n DNA. Nature 1988, 332:278-281. THEINSt., JEFFREYS aJ, GOOI He, COTrER F, FLINTJ, O'CONNOR NJT, \X/AINSCOATJS: Detection of Somatic Changes in H u m a n Cancer DNA by DNA Fingerprint Analysis. B r J Cancer 1987, 55:353-356. KELLYR, BULFIEkOG, COmCK A, GIBBS M, JEFFREYSAJ: Characterization of a Highly Unstable Mouse MinisateRite Locus: Evidence for Somatic Mutation during Early Development. Genomics 1989, 5:844-856.
KELLYR, GIBBS M, COLLICKA, JEFFREY'S AJ: Spontaneous Mutation at the Hypervariable Mouse MinisateUite Locus m s 6 hm: Flanking DNA Sequence and Analysis of Germline and Early Somatic Mutation Events. Proc R Soc Lond [Biol] 1991, 245:235-245. The mouse locus Msghm shows a high gernlline mutation rate, but also a high incidence of early somatic mutation, resulting in mice mosaic for mutant and non-mutant cells.This paper analyzes these mutation events, and reports a second m o u s e locus at which early somatic mutation appears to occur frequently.
66. .
GRAYIC, JEFFREYS AJ: Evolutionary Transience of Hypervariable Minisatellites in Man and t h e Primates. Proc R Soc Lond [Biol] 1991, 243:241-253. Two h~ervariable minisatellites in humans are shown to be short and monomorphic in other non-human primates. Thus the expansion of a locus into a highly unstable long tandemly repeated array is a process of great transience in evolution. The work includes a general discussion of minisatellite evolution. 67.
WOLFFRK, NAKAMURAY, WHITE R: Molecular Characterization of a Spontaneously Generated N e w Allele at a VNTR Locus: no Exchange of Flanking DNA Sequence. Genomics 1988, 3:347-351.
68.
WOLFFRK, PLAETKER, JEFFREYS AJ, WHITE R: Unequal Crossingover b e t w e e n Homologous C h r o m o s o m e s is not the Major Mechanism Involved in the Generation of New Alleles at VNTR Loci. Genomics 1989, 5:382-394.
69.
JARMANA, NICHOLLS RD, WEATHERALLDJ, CLEGG JB, HIGGS DR: Molecular Characterization of a Hypervariable Region Downstream of the H u m a n et-globin Gene Cluster. EMBO J 1986, 5:1857-1863.
70.
PAGEDe, BIEKER K, BROWN LG, HINTON S, LEPPERT M, L~d.OUEL J-M, La,THROP M, NYSTROM-LAHTIM, DE LA CI-U~.PPELkEA, WHITE R: Linkage, Physical Mapping and DNA Sequence Analysis of Pseudoautosomal Loci on the H u m a n X and Y Chromosomes. Genomics 1987, 1:243-256.
62. •
JEEFREYSAJ, NEUMANN R, WffSON V: Repeat Unit Sequence Variation in MinisateUites: a Novel Source of DNA Polymorphism for Studying Variation and Mutation by Single Molecule Analysis. Cell 1990, 60:473-485. The minisatellite D1S8 has alleles composed of an interspersed mixture of two repeat-unit t3T0es. This paper presents internal mapping data at this locus, demonstrates the polarity of variability, and shows that sizeselection and PCR can be used to recover individual deletion mutant molecules from bulk somatic or gemlline DNA.
71.
63. •.
64. •
ARMOURJAL, CROSIERM, JEFFREYSAJ: H u m a n Minisatellite Alleies Detectable only after PCR Amplification. Genomics 1992, 12:116-124. Demonstrates that some alleles at two minisateUite loci cannot be detected by standard Southern-blot hybridization. At one of these loci, the undetected allele has just three repeat units, and appears to be an intermediate in the evolutionary expansion of the locus from a monomeric state in non-human primates to larger numbers of repeats in most human alleles. 65. •,
CASKEY CT, PIT~UT1 A, FU Y-H, FENRVICK RG JR, NELSON DI. Triplet Mutations in H u m a n Disease. Science 1992, 256:784--789. A review of recent developments in the elucidation of diseases, such as myotonic dystrophy and fragile-X syndrome, associated with changes in the size of a trinucleotide array.
NAKAMURA Y, JULIER C, WOLFF R, HOLMT, O'CONNEt.L P, LEPPERT
M, WHITE R: Characterization of a H u m a n 'Midisatellite' Sequence. Nucleic Acids Res 1987, 15:2537-2547. 72. ••
WAHLSWP, WAI.~CE LJ, MOORE PD: Hypervariable Minisatellite DNA is a Hotspot for Homologous Recombination in H u m a n Cells. Cell 1990, 60:95-103. Minisatellite tandem repeats enhance plasmid-plasmid recombination in cultured cells. Although gene conversions also occur, the minisatellites mainly stimulate reciprocal exchanges.
73.
KROWCZYNSKAAM, RUDDERS RA, KRONTIRIS TG: The H u m a n MinisateUite C o n s e n s u s at Breakpoints of O n c o g e n e Translocations. Nucleic Acids Res 1990, 18:1121-1127.
74.
CH&NDLEYAC, MITCHELLAR: Hypervariable MinisateUite Regions are Sites for Crossing-over at Meiosis in Man. Cytc, genet Cell Genet 1988, 48:152-155.
75.
KOBORIJA, STRAUSS E, MINARD K, HOOD L: Molecular Analysis of the Hotspot of Recombination in the Murine Major Histocompatibility Complex. Science 1986, 234:173-179.
JAL Armour and AJ Jeffr~,s, Department of Genetics, University of Leicester, Leicester I.E1 7RH, UK.
Highly repetitive minisatellites' include the most variable human loci described to date. They have proved invaluable in a wide variety of genetic analyses, and despite some controversies surrounding their practical implementation, have been extensively adopted in civil and forensic casework. Molecular analysis of internal allelic structure has provided detailed insights into the repeat-unit turnover mechanisms operating in germline mutations, which are ultimately responsible for the extreme variability seen at these loci.
Current Opinion in Genetics and Development 1992, 2:850-856
Introduction Polymorphism in DNA structure provides the basis of genetic analysis. The first human DNA-sequence variants to be analyzed directly were base substitutional polymorphisms affecting the recognition sites for restriction enzymes [1]. As these restriction-site dimorphisms can only have two allelic states, their informativeness is limited to a ma.ximum heterozygosity of 50%. Following the first description of a highly polymorphic locus in human DNA [2], further examples of loci at which multi-allelic variability in allele length (Fig. 1) was caused by differences in the numbers of short repeated units were described
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[3-5]. Tandem repetition is widespread in the human and other eukaryotic genomes with variation in the number of tandemly repeated units occurring on many different scales. At one extreme, the alphoid satellites of human centromeres can have arrays up to 5 Mb in total length [6], and pulsed-field gel electrophoresis analysis has shown variation in the array length at different centromeres [7,8]. At the other end of the scale, arrays of dinucleotide repeats [9,10] and even mononucleotide repeats in retroposon tails [ 11 ] frequently show variation in the number of repeat units. Here, we will discuss the biology and applications of hypervariable 'minisatellites', at which total array size is generally in the range 0.5-30kb. The discovery and use of probes that are able to simultaneously detect large numbers of hypervariable tandemly repeated minisatellite loci have shown that they are widespread in the human genome [12], and have provided the basis for the systematic isolation of minisatellites by cloning [13-16,17,18oo].
Fig. 1. Schematic representation of the general principle of allele length variation caused by variation in tandem repeat copy number. Three different alleles are shown, containing five, eight or nine repeat units (black arrows). The consequent variation in the length of the array can be assayed as restriction fragment length variation by using Southern-blot hybridization after digestion with a restriction enzyme, such as X used here, that does not cut within the repeat array. Alternatively, relatively short arrays can be amplified using flanking single-copy primers, such as A and B (open arrows).
Minisatellites in genetic analysis Multi-locus DNA fingerprinting Using tandemly repeated probes in hybridization at low stringency, it has been demonstrated that highly variable tandemly repetitive loci are abundant in the hu-
Abbreviation MVR--minisatellite variant repeat.
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Biology and applications of human minisatellite loci Armour, Jeffreys 851 man genome [12]. The simultaneous detection of many hypervariable loci has also provided a powerful tool in genetic analysis. Because the combinatorial resolution of large numbers of highly polymorphic loci make the profile individual-specific, the profile is called the 'DNA fingerprint' or 'genetic fingerprint' [19]. In addition to these probes (ultimately derived from an intron in the human myoglobin gene; see [12]), other naturally occurring [20,21] and synthetic [22,23] tandem repeats have been successfully used to detect multiple hypervariable loci in human DNA. Individual-specific, multi-locus DNA fingerprints have a wide variety of applications, particularly in the establishment of family relationships [24,25 °] and to a lesser extent in forensic identification [26]. The high germline mutation rate at hypervariable minisatellite loci causes occasional anomalies in parentage analysis [12], but new mutations can almost always be distinguished with confidence from incorrect paternity ([25.]; see below). The obvious exception to individual specificity is in the case of monozygotic twins; indeed, the determination of zygosity in twins is an important special application of DNA fingerprinting [27]. In addition to their uses in human genetic analysis, multi-locus DNA fingerprints also have important applications in non-human species [28--31].
PCR typing at tandemly repeated loci An additional level of sensitivity can be achieved by using DNA amplification. PCR analysis of length variation at highly informative minisateUite loci is possible, but suffers from the disadvantage that many alleles at highly polymorphic loci are too large (e.g. 10-20kb) to amplify efficiently [40]. A few minisatellite loci combine high informativeness with restricted allele size, such that complete PCR profiles can be obtained [41%42]. The more abundant dinucleotide [9,10] and simple tandem repeat [43"*] loci can be typed by PCR to give phenotypes that, in principle, can be defined precisely by allele size; the small size of alleles (typically 100-200 bp) also means that they will be relatively resistant to DNA degradation in forensic specimens, as demonstrated by two extreme cases [44"',45"]. However, the informativeness of microsatellite loci is relatively limited, such that multiple loci often need to be typed to give results of significant statistical weight [44"]; furthermore, the systematic generation of series of artefactual 'slippage' products during the amplification of short repeat arrays [9,10] can introduce uncertainty in evidentiary use. PCR-based typing of highly variable minisatellites by sampling internal repeatunit variants (minisatellite variant repeat (lVlVR)-PCR; see below) has the advantages of sensitivity, extreme variability and digital encoding of phenotype, and can be applied to the analysis of partially degraded DNA [46.*].
Single locus profiles Hypervariable minisatellite loci studied singly, using cloned locus-specific probes in hybridization at high stringency, have a wide variety of applications as highly informative genetic markers. In segregation analysis, for example, analysis of linkage to a genetic disease is made more efficient by using extremely informative loci (for example, see [32]). Their use in general linkage analysis is however limited by their uneven distribution in the genome: while minisatellites appear to be approximately equally represented on all human autosomes, most hypervariable loci studied by in situ hybridization have been localized to subtelomeric regions [18",33,34]. Similarly, hypervariable loci mapped by linkage preferentially localize to near the ends of linkage maps [17,18",35,36]. Although similar clusters seem to exist in cattle [31], minisatellites do not appear to localize to subtelomeric regions in mice [37]. The high level of informativeness at many minisateUites nevertheless makes them very useful in a wide variety of genetic analyses. Their efficiency in distinguishing between the two alleles of an individual at a specific locus is of particular importance in detecting allele losses from tumours [38] and in the detection of karyotypic abnormalities such as uniparental disomy [39"]. In the deterruination of individual identity from forensic specimens, the use of single loci has the advantage that results can be obtained from less DNA than required for multi-locus DNA fingerprinting, and from DNA mixtures in forensic casework [14]; the DNA profiles obtained are also simpler to interpret, and can be databased as estimated allele lengths.
Controversies in evidentiary use Laboratory practice and data interpretation The use of minisateUite loci as evidence in legal proceedings, usually in the determination of paternity or individual identification in forensic medicine, has generated considerable debate on the reliability of DNA profiling in general and the use of minisatellite loci in particular [47]. While not placing undue emphasis on "exaggerated concerns over minor imperfections" [48], the potential problems in the evidentiary uses of minisatellite loci deserve discussion in a review of their applications. Many of the material criticisms of DNA profiling have centred on the results of relaxed laboratow practice, and of low or inconsistent standards of data interpretation - - primarily matters for standards of accreditation and quality control in the laboratories involved. Of particular concern has been the need to establish an objective set of criteria sufficient to declare a match between a forensic specimen and a suspect's DNA. The problem is that bands on an autoradiograph may appear to match, but can strictly only be said to be indistinguishable at the resolution afforded by the gel system used. Indeed, there are circumstances, for example with a badly contaminated forensic specimen, in which alleles may migrate to different distances on the gel, despite coming from the same person ('bandshift'; [49"]). Furthermore, the degree of precision with which the apparent match can be defined determines in part the statistical weight attached to the result; the tighter the 'window' within which bands from the two matching samples must lie, the more unlikely it is that the match is fortuitous.
852
Genomesand evolution At some loci, a relatively small number of discretely identifiable alleles differing by integral numbers of repeat units may, in principle, be assayed with precision [41,]; however, the existence of even uncommon repeat units of non-standard sizes or base composition can generate anomalous 'non-integral' alleles. The usual compromise made at loci with essentially continuous allele size distributions, such as most minisatellites used in DNA profiling, is to make the conservative declaration that the two samples match to within a certain distance, say 1 mm, on the gel. This assumption of imprecision can then be built into the statistical appraisal of the result (for example, see [25 °] ). An extension of this approach is the use of predefined size intervals or 'bins' into which alleles are classified ([50"]; but see also [51]).
Mutation and population substructuring In addition to the issues of good laboratory practice and primary data interpretation raised above, the use of minisatellite profiling as evidence may be susceptible to two important sources of possible (non-human) error: gross variation in allele frequencies between subpopulations, and, in paternity disputes, germline mutation [52]. There is a theoretical possibility that mosaicism for a somatic mutation may lead to discrepancies in individual identification, but this would act to produce a false exclusion rather than a spurious inclusion [53]. The potential influence of population substmcturing derives from an assumption that the tendency of germline mutation to diversify minisatellite alleles is significantly counteracted by the tendency of genetic drift to fix alleles within a subpopulation. Thus, population substructure might lead to a particular DNA profile being less rare within, for example, an ethnic minority or genetically isolated population than in the larger population from which the allele-frequency estimates were derived. Empirical analysis of multi-locus DNA fingerprints, based on extensive paternity casework, suggests that paternity can be established with confidence even in the presence of germline mutations [25"]; furthermore, at least within the (Caucasian) populations studied, population substructuring does not appear to exert significant influence on the statistical weight of results. Although a number of studies have directly addressed this issue (for example, [54] ), the empirical evidence is much less clear for minisatellite loci examined singly, and the extent to which population heterogeneity influences the statistical confidence of DNA profiling results has been a matter of dispute [55-.,56.o,57.]. The extremely (some would say excessively) conservative approach suggested by Lewontin and Hard [55"'] includes the need for databases appropriate to each ethnic group, and the empirical estimation of probabilities that make no assumptions about the population genetics of minisatellite loci. For example, a phenotype not hitherto encountered in a database of N individuals would have its frequency conservatively estimated as 1/N [55"']. These population genetic issues, as well as specific recommendations for conservative approaches to the estimation of genotype frequencies from
allele frequencies, have recently been discussed in a National Academy report on DNA technology in forensic science [58°]. The practical difficulty of such a conservative approach, namely the need to assemble large databases, based on error-prone allele-length measurement, for each locus and each ethnic group, may in principle be mitigated by the adoption of digital phenotyping systems such as MVRPCR [46..]. A rapid typing system is used in MVR-PCR to assay a second level of variability within minisatellite arrays, the distribution of different types of repeat unit along an allele (see below and Fig. 2). Genomic DNA is typed directly, to give a diploid profile resulting from the superimposition of the two allelic patterns. These extraordinarily variable, digitally encoded phenotypes have the advantage that the data are unambiguous, providing simple match criteria, and are 'portable' - - there is no need for 'side by side' comparison to establish identity or exclusion, and data can be exchanged between centres, allowing the cumulative assembly of large phenotypic databases.
Length-change mutation and the origin of variation General parameters of the mutation process Allelic diversity at minisatellite loci is generated by a high rate of spontaneous germline mutation to new-length alleles. At extremely variable loci, germline mutations to alleles containing new numbers of repeat units occur at a rate high enough to measure by direct observation in pedigree analysis [59]. The observed mutation rates to new-length alleles correlate with heterozygosity as predicted by a model of neutral mutation and random drift. While at most loci mutations appear to arise with similar frequency in male and female germlines, at least one locus shows a predominance of mutations originating in the male germline [18,.]. Although germline mutation is the ultimate source of allelic variability in populations, somatic mutations can also occur at minisatellite loci [53,60]. Somatic instability is a prominent feature of two mouse minisatellites [61,62.], at which somatic mutation early in development gives rise to mice globally mosaic for mutant and non-mutant cells. Somatic deletion mutants at the minisatellite D1S8 (MS32) in humans have also been recovered from blood DNA by using size-selection followed by single molecule dilution and PCR [63°']. The apparent relative stability in the population of small minisatellite alleles provides indirect evidence that the germline mutation process may operate at a frequency dependent on allele length [64.]. Similarly, the threshold effects seen at trinucleotide arrays associated with the fragile-X syndrome and myotonic dystrophy [65"] also suggest a strong correlation between allele length and mutation rate for these disease loci. Analysis of cognate minisatellite loci in non-human primates likewise suggests that short arrays can be stable over millions of years [66°].
Biology and applications of human minisatellite loci Armour, Jeffreys
(a)
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Fig. 2. Minisatellite variant repeat (MVR) mapping. Many minisatellite loci have alleles composed of an interspersed mixture of two or more different types of repeat unit. In this example, the two repeat-unit types are distinguished by shading. The pattern of interspersion of the repeat-unit types (the 'internal map' or 'MVR map') can in some cases be assayed using restriction mapping. (a) Enzyme E2 has sites (indicated by vertical bars) in both types of repeat; however, only the shaded variant repeat contains a site for enzyme El. Thus, mapping sites for El can be used to map the positions of a particular repeat-unit type. (b) A more efficient method, MVR-PCR, uses PCR primers to discriminate between repeat-unit types. MVR-map variation represents a second level of variation within minisatellite alleles: for example, the two alleles shown below have the same number of repeat units (19), but their internal maps show that they are clearly distinct alleles. Thus, an individual with these two alleles would appear to be a homozygote for allele length, but is in fact heterozygous, as shown by MVR mapping. Digital DNA typing uses MVR-PCR to analyze both alleles simultaneously to generate the digital code indicated: I, both repeats unshaded; 2, both shaded; and 3, heterozygous.
The mechanisms by which minisatellite-length changes may occur include replication slippage, intramolecular recombination, unequal sister-chromatid exchange, and unequal inter-allelic recombination or gene conversion. The approximately equal frequency of mutations increasing or decreasing allele length excludes intramolecular recombination as the predominant mechanism [59]. Analysis of a single germline mutation at D17S5 (YNZ22) showed that polymorphisms flanking the minisatellite were not exchanged during the mutation process, thus excluding a simple recombinational model for that event [67]. Similarly, analysis of a series of mutations at DIS7 (MS1) failed to show any increase in recombination between distal flanking markers associated with mutation
[68].
Internal m a p p i n g of minisatellite
alleles
More detailed analysis of minisatellite variation and mutation has been possible by studying a second level of variation at minisatellite loci. In addition to allelic variation in the number of tandem repeats, all minisatellites that have been extensively studied show subtle variations in the precise sequence of repeat units [12-14,69-71], such that a minisatellite allele is frequently composed of an interspersed mixture of two or more MVRs (Fig. 2) [46°.,63°.]. The patterns of interspersion of variant repeats were initially ascertained using restriction-site mapping [63o°], but MVR-PCR has recently provided a much more rapid method for MVR mapping [46o°].
Studies of the internal disposition of variant repeats within alleles shows that there is variation in internal map structure as well as in length; indeed, internal map variation can be truly enormous, with perhaps as many as 108 different alleles at D1S8 in the present world population [46°°]. More surprisingly, there is a gradient of variability; at one end of the locus, the pattern of repeat units shows only a small number of distinct types in different alleles, while the other end is 'ultravariable', with nearly each allele having its own unique pattern. While this polarity was detected first in the MS32 (D1S8) minisatellite [46°,,63°°], there is evidence for similar gradients of variability at two other loci (JAL Armour, DL Neff and AJ Jeffreys, unpublished data). This preferential localization of variability at one end of minisatellite loci predicts that germline mutations would be localized at. this end of the array, and analysis of germline mutations at D1S8 and other minisateUite loci shows that germline mutation events are indeed concentrated in the first few repeats at the 'ultmvariable' end [46 °°] (JAL Armour and AJ Jeffreys, unpublished data). This region, then, appears to be a hot-spot for length-change mutations, and suggests that the mutation process at these loci may be under the influence of some as yet unidentified c/s-acting element. By comparing the maps of new mutant and progenitor alleles, internal mapping of minisateUite structure also allows conjectures to be made about the mechanisms of minisateUite mutation. This more recent work shows that; while the majority of mutation events appear to be entirely intra-allelic, some events can only be satisfactorily explained as the result of inter-aUelic exchanges between
853
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Genomes and evolution
parental alleles. This observation may parallel other evidence implicating minisatellites in recombinational processes and in genomic instability [33,72.o,73-75], and suggests further that the nautational hot-spot at one end of these arrays may also be a hot-spot for meiotic recombination and/or gene conversion [46-. ] (JAL Armour and AJ Jeffreys, unpublished work).
the Cardiac Muscle Actin Gene. A m J H u m Genet 1989, 44:397-401. 11.
ECONOMOUEP, BERGENAW, WARRENAC, ANTONARAKISSE: The Polydeoxyadenylate Tract of Alu Repetitive Elements is Polymorphic in the Human Genome. Proc Nail Acad Sci USA 1990, 87:2951-2954.
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JEFFI~WSAJ, WILSON V, THEIN SL: Hypervariable 'Minisatellite' Regions in Human DNA. Nature 1985, 314:67-73.
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\VONGZ, WILSONV, JEFFREYSAJ, THEIN Sl2 Cloning a Selected Fragment from a Human DNA 'Fingerprint': Isolation of an Extremely Polymorphic Minisateliite. Nucleic Acids Res 1986, 14:4605-4616.
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WONGZ, WILSON V, PATEI. I, pO\rEy S, JEFFREYS AJ: Characterization of a Panel of Highly Variable Minisatellites Cloned from Human DNA A n n H u m Genet 1987, 51:269-288.
15.
NAKAMURAY, LEPPERT M, O'CONNELL P, WOLFF R, HOhM T, CUL\q?.R M, MARTINC, FUJIMOTO E, HUFF M, KUMIJN E, WHITE R: Variable Number of Tandem Repeat (VNTR) Markers for Human Gene Mapping. Science 1987, 235:1616-1622.
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NAKAMURAY, CARLSONM, KRAPCHOK, KANAMOmM, WHITE R: New Approach for Isolation of VNTR Markers. A m J H u m Genet 1988, 43:854-859.
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ARMOURJAL, POVEY S, JEREMIAH S, JEFFREYS AJ: Systematic Cloning of Human Minisateilites from Ordered Array Charomid Libraries. Genomics 1990, 8:501-512.
Conclusion
The extension of minisatellite profiling to MVR analysis promises a wealth of detailed information on their applications in genetic analysis, including evidentiary uses, as well as in the elucidation of mutational mechanisms.
Acknowledgements We thank D Monckton for helpful comments on time manuscript. JAL Armour is a Wellcome Trust postdoctoral fellow. Work carried out in AJ Jeffreys group is supported by grants from tile MRC and the Royal Society.
VERGNAUDG, MARIAT D, APIOU F, AURIAS A, LATHROP M, LAUTHIERV: The Use of Synthetic Tandem Repeats to Isolate New VNTR Loci: Cloning of a Human Hypermutable Sequence. Genomics 1991, 11:135-144. Describes the isolation of hypervariable loci from human DNA by hybridization with syntheUc tandem repeats. One of the loci isolated is extremely unstable; mutation occurs preferenUally in timemale gemmline, with a mutation rate to new-length alleles of 15% per spemm. 18. ida
References and recommended reading Papers of particular interest, published within the annual period of mview, have been highlighted as: • of special interest •. of outstanding interest
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FOWLERSJ, GIlL P, WERRETr DJ, HIGGS DR: Individual Specific DNA Fingerprints from a Hypervariable Region Probe: Alpha Globin 3'HVR. H u m Genet 1988, 79:142-146.
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JEFFREYSAJ, BROOKFIELDJFY, SEMEONOFF R: Positive Identification of an Immigration Test-case Using Human DNA Fingerprints. Nature 1985, 317:818-819.
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7.
OAr~-¢ R, "I'¢~R-SMITH C: Y Chromosome DNA Haplotyping Suggests that Most European and Asian Men are Descended from One of Two Males. Genomics 1990, 7:325-330.
8.
MAHTANI MM, WILIARDHF: Pulsed-field Gel Analysis o f Alpha Satellite DNA at the Human X Chromosome Centromere: High Frequency Polymorphisms and Array Size Estimate. Genomics 1990, 7:607-613.
25. •
JEFFREYSAJ, TURNER M, DEBENHAMP: The Efficiency of Multilocus DNA Fingerprint Probes for Individualization and Establishment of Family Relationships, Determined from Extensive Casework. Am J H u m Genet 1991, 48:824-4340. The results of over 1700 paternity cases analyzed using DNA fingerprinting are examined. The data provide empirical evidence for the reliability of DNA fingerprinting in casework, and the authors address the issue of mutation in the assessment of paternity. 26.
GILL P, JEFFREYS AJ, WERRE'Vr DJ: Forensic Applications of DNA 'Fingerprints'. Nature 1985, 318:577-579.
9.
WEBERJL, MAY PE: Abundant Class of Human DNA Polymorphisms which Can be Typed Using the Polymerase Chain Reaction. A m J H u m Genet 1989, 44:388-396.
27.
HILL AVS, JEFFREYS AJ: Use of Minisatellite DNA Probes for Determination o f Twin Zygosity at Birth. Lancet 1985, ii:1394--1395.
10.
Ll'r'r M, LUTY JA: A Hypervariable Microsatellite Revealed by in Vitro Amplification o f a Nucleotide Repeat within
28.
JEFFREYSAJ, WILSON V, KELLYR, TAYLORB, BUt.FIELDG: Mouse DNA 'Fingerprints': Analysis of Chromosome Localization
Biology and applications of human minisatellite loci Armour, Jeffreys and Germ-line Stability of Hypervariable Loci in Recombinant Inbred Strains. Nucleic Acids Res 1987, 15:2823-2836. 29.
JEFFREYSAJ, MORTON DB: DNA Fingerprints of Dogs and Cats. Animal Genet 1987, 18:1-15.
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BURKET, BRUFORDMW': DNA Fingerprinting in Birds. Nature 1987, 327:149-152.
31.
GEORGES M, LATHROP M, l-fILBERT P, MARCO'ITE A, SCHWERS A, SWILLENS S, VASSART G, HANSET R: On the Use of DNA Fingerprints for Linkage Studies in Cattle. Genomics 1990,
6:461-474. 32.
REEDERSST, BREUNING MH, DAVIES KE, NICHOLLS RD, JARMAN AP, HIGGS DR, PEARSON PL, WEATI-IERALLDJ: A Highly Polymorphic DNA Marker Linked to Adult Polycystic Kidney Disease on C h r o m o s o m e 16. Nature 1985, 317:542-544.
33.
ROYLENJ, CLARKSONRE, \X/ONG Z, JEFFREYS AJ: Clustering of Hypervariable MinisateUites in the Proterminal Regions of H u m a n Autosomes. G e n o m i ~ 1988, 3:352-360.
34.
ARMOURJAL, WONG Z, WILSON V, ROYLE NJ, JEFFREYS AJ: Sequences Flanking the Repeat Arrays of H u m a n Minisatellites: Association with T a n d e m and Dispersed Repeat Elements. Nucleic Acids Res. 1989, 17:4925-4935.
35.
NAKAMURAY, LAT]-IROPM, O'CONNELI. P,LEPPERT M, LALOUELjM, WHITE R: A Primary Map of T e n DNA Markers and T w o Serological Markers for H u m a n C h r o m o s o m e 19. Genomics 1988, 3:67-71.
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LATHROP GM, NAKAMURA Y, O'CONNELL P, LEPPERT M, WOODWARD S, LALOOEI.J-M, WHITE R: A Mapped Set of Genetic Markers for H u m a n C h r o m o s o m e 9. Genomic.~ 1988, 3:361-366.
Describes the analysis of DNA taken from an eight year old body. Although DNA extracted from skeletal remains was badly degraded, sufficient remained to allow identification by comparison with the presumed victim's parents at short dinucleotide (microsatellite) loci. 45. •
JEFFREYSAJ, ALLENM, HAGELBERG E, SONNBERG A: Identification of the Skeletal Remains of Josef Mengele by DNA Analysis. Forensic Sci Int 1992, 56:65-76. PCR of microsatellite loci solves the mystery of the Auschwitz 'Angel of Death'.
46. .•
JEFFREYSAJ, MACLEOD A, TAMAKIK, NEIL DL, MONCKTON DG: MinisateUite Repeat Coding as a Digital Approach to DNA Typing. Nature 1991, 354:204-209. The MVR-PCR technique described here is used to analyze variation in the pattern of interspersion of two repeat-unit types at the minisatellite D1S8. The results can be encoded in a digital form, and provide a rapid assay both for individual identity in forensics and in the analysis of repeat-unit turnover in minisatellite evolution. 47.
LANDER ES: DNA Fingerprinting on Trial. Nature 1989, 339:501-505.
48.
KOSHLANDDE JR: Editorial: DNA Fingerprinting and Eyewitness Testimony. Science 1992, 256:593.
49. •
THOMPSONWC, FORD S: T h e Meaning of a Match: Sources of Ambiguity in the Interpretation of DNA Prints. In Forensic DNA Tedmology. Edited by Farley MA and Harrington JJ. Chelsea, Michigan: Lewis Publishers, Inc.; 1991:93-152. Detailed discussion of the practical problems arising in the interpretation of forensic DNA profiles. 50. •
BUDOWLEB, GIUSTI AM, WAYEJS, BAECHTELFS, FOURNEY RM, ADAMS DE, PRESI.EY LA, DEADMAN HA, MONSON KL: Fixed-Bin Analysis for Statistical Evaluation of C o n t i n u o u s Distributions of Allelic Data from VNTR Loci, for Use in Forensic Comparisons. Am J Hztm Genet 1991, 48:841-855. Develops a conservative approach to the statistical evaluation of matches at single minisatellite loci; the presence or absence of an allele in one of a predefined series of gel intervals, or size "bins', is the basic parameter used.
37.
JUUERC, DE GOUYON B, GEORGES M, GUENET J-L, NAKAMURA Y, AVNER P, LATHROP M: Minisatellite Linkage Maps in the Mouse by Cross-Hybridization with H u m a n Probes Containing T a n d e m Repeats. Proc Nail Aca d Sci USA 1990, 87:4585-4589.
38.
VOGEl.STEINB, FEARON ER, KERN SE, HAMILTONSR, PREISINGER AC, NAKAMURAY, WHITE R: Allelotype of Colorectal Carcinomas. Science 1989, 244:207-211.
51.
LANDERES: Invited Editorial: Research on DNA Typing Catching Up with Courtroom Application. A m J H u m Genet 1991, 48:819-823.
MALCOLM S, CLAYTON-SMITH J, NICHOLS M, ROBB S, WEBB T, ARMOURJAL, JEFI:REYS AJ, PEMBREY ME: Uniparental Paternal Isodisomy in Angelman's Syndrome. Lancet 1991, 337:694-697. The exclusively paternal inheritance of both copies of c h r o m o s o m e 15 in some cases of Angelman's syndrome is clearly demonstrated using a hypervariable locus.
52.
COHENJE: DNA Fingerprinting for Forensic Identification: Potential Effects on Data Interpretation of Subpopulation Heterogeneity and Band N u m b e r Variability. A m J H u m Genet 1990, 46:358-368.
53.
ARMOURJAL, PATEL 1, THEIN SL, FEY M, JEFFREYS AJ: Analysis of Somatic Mutations at H u m a n MinisateUite Loci in T u m o u r s and Cell Lines. Genomics 1989, 4:328-334.
54.
DEVLIN B, RISCH N, ROEDER K: No Excess of Homozygosity at Loci Used for DNA Fingerprinting. Science 1990, 249:1416--1420.
39. •
40.
JEFFREYSAJ, WIL¢~ONV, NEUMANNR, KEY'rEJ: Amplification of H u m a n Minisatellites by the Polymerase Chain Reaction: Towards DNA Fingerprinting of Single Cells. Nucleic Acids Res 1988, 16:10953-10971.
41.
BUDOWLEB, CHAKRABORTYR, GIUSTI AM, EISENBERG AJ, ALLEN RC: Analysis of the VNTR Locus D1SSO by the PCR Followed by High-Resolution PAGE. Am J H u m Genet 1991, 48:137-144. Describes the PCR typing of a highly informative locus to give discretely classifiable alleles. .
42.
BOERWINKLEE, XIONG W, FOOREST E, CHAN L: Rapid Typing of Tandemly Repeated Hypervariable Loci by the Polymerase Chain Reaction: Application to the Apolipoprotein B 3' Hypervariable Region. P m c Nail Ac ad Sci USA 1989, 86:212-216.
EDWARDSA, CmTELLO A, HAMMONDHA, CASKEYCT: DNA Typing and Genetic Mapping with Trimeric and Tetrameric T a n d e m Repeats Am J H u m Genet 1991, 49:746-756. Demonstrates the utility of arrays composed of tri- or tetranucleotide repeats as informative multiallelic genetic markers.
43. °.
44. .°
HAGELBERGE, GRAY IC, JEFFREYS AJ: Identification of the Skeletal Remains of a Murder Victim by DNA Analysis. Nature 1991, 352:427-429.
55.
LEWOr, mN RC, HARTL DL: Population Genetics in Forensic DNA Typing. Science 1991, 254:1745-1750. his work details the population genetic basis for reservations about some current practices in the forensic use of minisatellite typing. 56. CHAKRABORTYR, KJDD KK: T h e Utility of DNA typing in .• Forensic Work. Science 1991, 254:1735-1739. An answer to some of the points raised by Lewontin and Hard [55°°]. The (controversial) circumstances surrounding the publication of this article are discussed in the News a n d Comment section of this same issue of Science. Correspondence arising from this, and from Lewontin and Hard's article, appear in the 28 February 1992 issue of Science. See
also [57"]. 57.
BROOKFmLD J: Law and Probabilities. Nature 1992, 355:207-208. Summarizes the Lewontin and Harfl [55 °°] vs Chakrabony and Kidd [56 *° ] debate regarding DNA typing in forensic law, and describes the surrounding controversy. ,
58, w
NATIONALRESEARCHCOUNCIL,COMMITTEEON DNA TECHNOLOGY IN FORENSIC SCIENCE (McKuSlCK VA, CHAIRMAN):DNA Technol-
855
856
Genomes and evolution ogy in Forensic Science. Washington DC: National Academy Press; 1992. A comprehensive review of many aspects of the use of DNA typing in forensic studies. The report endorses the general use of DNA typing in forensic science, and makes specific recommendations for conservative approaches to the statistical evaluation of typing results.
59.
60.
61.
JEFFREYS AJ, ROYLE NJ, WILSON V, WONG Z: Spontaneous Mutation Rates to New Length Alleles at Tandem-repetitive Hypervariable Loci in H u m a n DNA. Nature 1988, 332:278-281. THEINSt., JEFFREYS aJ, GOOI He, COTrER F, FLINTJ, O'CONNOR NJT, \X/AINSCOATJS: Detection of Somatic Changes in H u m a n Cancer DNA by DNA Fingerprint Analysis. B r J Cancer 1987, 55:353-356. KELLYR, BULFIEkOG, COmCK A, GIBBS M, JEFFREYSAJ: Characterization of a Highly Unstable Mouse MinisateRite Locus: Evidence for Somatic Mutation during Early Development. Genomics 1989, 5:844-856.
KELLYR, GIBBS M, COLLICKA, JEFFREY'S AJ: Spontaneous Mutation at the Hypervariable Mouse MinisateUite Locus m s 6 hm: Flanking DNA Sequence and Analysis of Germline and Early Somatic Mutation Events. Proc R Soc Lond [Biol] 1991, 245:235-245. The mouse locus Msghm shows a high gernlline mutation rate, but also a high incidence of early somatic mutation, resulting in mice mosaic for mutant and non-mutant cells.This paper analyzes these mutation events, and reports a second m o u s e locus at which early somatic mutation appears to occur frequently.
66. .
GRAYIC, JEFFREYS AJ: Evolutionary Transience of Hypervariable Minisatellites in Man and t h e Primates. Proc R Soc Lond [Biol] 1991, 243:241-253. Two h~ervariable minisatellites in humans are shown to be short and monomorphic in other non-human primates. Thus the expansion of a locus into a highly unstable long tandemly repeated array is a process of great transience in evolution. The work includes a general discussion of minisatellite evolution. 67.
WOLFFRK, NAKAMURAY, WHITE R: Molecular Characterization of a Spontaneously Generated N e w Allele at a VNTR Locus: no Exchange of Flanking DNA Sequence. Genomics 1988, 3:347-351.
68.
WOLFFRK, PLAETKER, JEFFREYS AJ, WHITE R: Unequal Crossingover b e t w e e n Homologous C h r o m o s o m e s is not the Major Mechanism Involved in the Generation of New Alleles at VNTR Loci. Genomics 1989, 5:382-394.
69.
JARMANA, NICHOLLS RD, WEATHERALLDJ, CLEGG JB, HIGGS DR: Molecular Characterization of a Hypervariable Region Downstream of the H u m a n et-globin Gene Cluster. EMBO J 1986, 5:1857-1863.
70.
PAGEDe, BIEKER K, BROWN LG, HINTON S, LEPPERT M, L~d.OUEL J-M, La,THROP M, NYSTROM-LAHTIM, DE LA CI-U~.PPELkEA, WHITE R: Linkage, Physical Mapping and DNA Sequence Analysis of Pseudoautosomal Loci on the H u m a n X and Y Chromosomes. Genomics 1987, 1:243-256.
62. •
JEEFREYSAJ, NEUMANN R, WffSON V: Repeat Unit Sequence Variation in MinisateUites: a Novel Source of DNA Polymorphism for Studying Variation and Mutation by Single Molecule Analysis. Cell 1990, 60:473-485. The minisatellite D1S8 has alleles composed of an interspersed mixture of two repeat-unit t3T0es. This paper presents internal mapping data at this locus, demonstrates the polarity of variability, and shows that sizeselection and PCR can be used to recover individual deletion mutant molecules from bulk somatic or gemlline DNA.
71.
63. •.
64. •
ARMOURJAL, CROSIERM, JEFFREYSAJ: H u m a n Minisatellite Alleies Detectable only after PCR Amplification. Genomics 1992, 12:116-124. Demonstrates that some alleles at two minisateUite loci cannot be detected by standard Southern-blot hybridization. At one of these loci, the undetected allele has just three repeat units, and appears to be an intermediate in the evolutionary expansion of the locus from a monomeric state in non-human primates to larger numbers of repeats in most human alleles. 65. •,
CASKEY CT, PIT~UT1 A, FU Y-H, FENRVICK RG JR, NELSON DI. Triplet Mutations in H u m a n Disease. Science 1992, 256:784--789. A review of recent developments in the elucidation of diseases, such as myotonic dystrophy and fragile-X syndrome, associated with changes in the size of a trinucleotide array.
NAKAMURA Y, JULIER C, WOLFF R, HOLMT, O'CONNEt.L P, LEPPERT
M, WHITE R: Characterization of a H u m a n 'Midisatellite' Sequence. Nucleic Acids Res 1987, 15:2537-2547. 72. ••
WAHLSWP, WAI.~CE LJ, MOORE PD: Hypervariable Minisatellite DNA is a Hotspot for Homologous Recombination in H u m a n Cells. Cell 1990, 60:95-103. Minisatellite tandem repeats enhance plasmid-plasmid recombination in cultured cells. Although gene conversions also occur, the minisatellites mainly stimulate reciprocal exchanges.
73.
KROWCZYNSKAAM, RUDDERS RA, KRONTIRIS TG: The H u m a n MinisateUite C o n s e n s u s at Breakpoints of O n c o g e n e Translocations. Nucleic Acids Res 1990, 18:1121-1127.
74.
CH&NDLEYAC, MITCHELLAR: Hypervariable MinisateUite Regions are Sites for Crossing-over at Meiosis in Man. Cytc, genet Cell Genet 1988, 48:152-155.
75.
KOBORIJA, STRAUSS E, MINARD K, HOOD L: Molecular Analysis of the Hotspot of Recombination in the Murine Major Histocompatibility Complex. Science 1986, 234:173-179.
JAL Armour and AJ Jeffr~,s, Department of Genetics, University of Leicester, Leicester I.E1 7RH, UK.