- Email: [email protected]
DNA fingerprinting: A new dimension
TREE vol. 5, no.
2 Caraco, T. (1980) Ecology 61, 119-128 3 Real, L.A. and Caraco, T. (1986) Annu. Rev.
Ecol.
Syst.
17, 371-390
4 Stephens, D.W. (1981) Anim.
Behav.
29,628-629
5 Houston, A.I. and McNamara, J.M. (1982) Anim. Behav. 30,1260-l 261 6 McNamara, J.M. (1984) SIAM J. Control
Opt/m.
2,87-94
7 Houston, A.I. and McNamara, J.M.
(1985) Behav. Ecol. Sociobiol. 17, 136-141 8 McNamara, J.M. and Houston, A.I. (1990) in Living in a Patchy Environment
5,
May 1990
11 Mazur, J.E. (1986) J. Exp. Psycho/. Anim.
Behav.
Processes
12,116-124
(Swingland, I. and Shorrocks, B., eds), pp. 23-43, Oxford University Press 9 Caraco, T., Martindale, S. and Whittam, T.S. (1980) Aim. Behav. 28,820-831 10 Herrnstein, R.J. (1964) J. fxp. Anal.
12 Staddon, J.E.R. and Reid, A.K. (1987) in Foraging Behavior (Kamil, J.R., Krebs, J.R. and Pulliam, H.R., eds), pp. 497-524, Plenum 13 Caraco, T., Blanckenhorn, W.U., Gregory, G.M., Newman, J.A., Recer, G.M. and Zwicker, S. (1990) Aim.
Behav.
Behav.
7,179-182
39,338-345
DNA Fingerprinting: ANewDimension JosephinePembertonand Bill Amos DONE IT AGAIN! Those friendly people who brought you DNA fingerprinting have now revealed genetic variation on an extraordinarily fine scale. Not only that: in a recent paper describing DNA sequence differences between alleles at a minisatellite (fingerprint) locus, Alec Jeffreys, Rita Neumann and Victoria Wilson of Leicester University, UK, have also answered fundamental questions about the rate and mode of generation of this variation’. DNA fingerprints2z3, those unevenly spaced ladders of bands, which in many species are unique for an individual, are the result of screening several extremely variable loci simultaneously. Each band represents an allele at a particular locus and is thus one of a pair. Its partner is usually elsewhere in the fingerprint profile (heterozygous state) but may occasionally occur at the same point (homozygous state). The DNA sequences detected by fingerprint probes are very short (e.g. 16 base pairs for the Jeffreys probe h33.15) and often occur in strings, known as tandem repeat arrays or minisatellite DNA. Variation in allele position on a fingerprint profile is due to differences in the length of the DNA fragments detected, which in turn result from variation in the number of repeats within an array. At minisatellite loci, repeat units appear to be easily gained or lost and different alleles can have anything from a handful to hundreds of repeats. It is this variation in repeat-unit number that causes the variation seen in DNA fingerprints. Although DNAfingerprintsare useful for determining close kin, for many other purposes (e.g. population surveys) it is useful to have locus-specific information. Unfortunately, sorting out which bands are THEY’VE
Josephine Pemberton and Bill Amos are at the Dept of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
132
allelic to one another in a DNA fingerprint is extremely difficult. The secret lies in finding conditions under which the DNA probe sticks to alleles at one locus alone, thus stripping away the confusing plethora of bands from other loci. This is achieved by first cloning, and then probing with, the DNA sequence specific to one locus, instead of using a generic minisatellite probe. The Leicester group is one of the few to have succeeded in this task. For a series of human single-locus fingerprint probes they have found locus-specific heterozygosities of 90-99% (Refs 4, 5). The latest paper’ takes this work
into a new dimension. From the outset it was clear that the repeat units within an allele, although very similar, do show subtle variations in sequence. These sequence variants presumably arise initially by point mutation, but since repeat units may share the same mutatior?, other processes must spread them between repeat units. To score such variation, one could sequence entire alleles, but the Leicester team avoided this brainnumbing exercise with an elegant ruse. Alleles at the human minisatellite locus that they investigated, called DlS8 (detected by probe MS32), have a repeat unit that is 29 base pairs long. All repeat units contain a recognition sequence for the restriction enzyme Hinfl. However, some repeat units-but not all - have
Allele A
Allele B
* 1 1L 1 1,! lw! 1 I* ! I*! 1 1
l l . l
Hid
*
*l *-
*_______--____-------------l _____-____-------------l ______----mmwm--*____---mmmmmm l _ _ _ _ -
_ -
Hinfl
H&II
Hi&
Had11
_--------__
--
------
-------_-_-
------
------
__----
Fig. 1. Schematic representation of how the internal mapping technique developed by Jeffreys eta/. can distinguish two alleles, A and 6. Top panel: Alleles are purified and radioactively labelled at one end 1”). While all repeat units in both A and B have a Hinfl restriction site (unbroken arrows), only a proportion also have a Haelll site (broken arrows). Middle panel: Partial digestion with Hinfl gives a population of end-labelled DNA fragments with one size class for each repeat unit. Partial digestion with Haelll gives fragments for only those repeat units with Haelll sites. Bottom panel: When electrophoresed and exposed to X-ray film, the Hinfl partial digests give complete ladders of bands, while the Haelll partial digest ladders have gaps. The precise position of the Haelll sites is readily determined by reference to the Hinfl ladder. Alleles A and 6, though having the same number of repeat units, are clearly distinguishable on the basis of their Haelll ladders.
TREE vol. 5, no. 5, May 7990
9
14
8
FHFHFHFH an additional recognition sequence, this time for the restriction enzyme Haelll. The beauty of the new work lies in the way in which this second source of variation is detected and read. After the polymerase chain reaction (PCR) had been used to make many exact copies of the DNA sequence of a particular allele, each molecule was radioactively labelled at one end and then aliquots were subjected to partial digestion with either Hinfl or Haelll. In a partial digest, insufficient restriction enzyme and/or time is allowed for the DNA to be cut at all recognition sites. Under the correct conditions, a population of DNA molecules is generated in which each possible cutting site is represented by end-labelled fragments of a discrete length (Fig. 1). When the products of a Hinfl partial digest are run on a gel, the result is an evenly spaced ladder of bands increasing in size by one repeat unit at a time. In contrast, the products of a Haelll partial digest result in an incomplete ladderthat lacks rungs because some repeat units are not cut by Haelll. If the two samples are run next to each other, the precise position within an allele of repeat units lacking a Haelll site can be mapped with reference to the Hinfl ladder of bands (Figs 1 and 2). Hence, each allele can be described by a binary internal map indicating the distribution of Haelll sites along it. The extra variability revealed by this technique is no less staggering than that revealed in the first DNA fingerprints. Among 32 DlS8 alleles amplified from humans of various origins and internally mapped, only two identical alleles were found. Importantly, alleles so similar in length that they are indistinguishable on a conventional fingerprint gel can have very different internal structures. Combining length variation with internal structure variability, Jeffreys eta/, calculate that for this locus alone they are now in a position to detect about 1070 different character states, should they exist! An important application of this technique will be in individual identification of criminals. Conventional DNA fingerprinting has already revolutionized forensic research6f7, but it does have an Achilles heel. For a host of reasons, not all gels run perfectly straight and it is sometimes extremely difficult to decide if two bands are identical or not. For example, in a recent double murder case in the Bronx, New York, DNA fingerprint evidence was disallowed as a result of a challenge to an interpretation that bands on a gel matched*. In future, lawyers will,
9
16 F
14 H
F
8
16
HFHFH
Fig. 2. Examples of internal mapping of four alleles (numbers 9,14,8,16) at DlS8. Each allele is represented by the gel electrophoresis pattern of a Hinfl partial digest IF) and a Haelll partial digest (H). Samples were electrophoresed on a standard gel (left) and an extension gel (above) to give maximum resolution throughout each allele. Reproduced with permission from Ref. 7.
rightly, be on the alert for such problems. The new technique not only increases accuracy by giving an exact measure of the length of an allele (from the number of repeat units detected by the Hinfl digest), but also gives a precise binary description of repeat unit types (from the Haelll digest). Furthermore, in contrast to conventional DNA fingerprints, the information is in a perfect format for storage in a computer database. It will surprise no-one to learn that the technique is the subject of patent applications. Comparing the internal maps of the 31 identified alleles, Jeffreys et al.’ observed distinct similarities
between certain alleles, including alleles of quite different lengths, especially at their 5’ ends. On the basis ofthese similarities, the authors identify four distinct groups, of which two (called 2A and 28) look closely related. This immediately raises the question of whether DlS8 alleles could be used to investigate the genetic history of the human race, shedding light on the growth and movement of peoples in the same way that mitochondrial DNA9 and other variable DNA regionslo have been applied. Whether internal mapping of fingerprint alleles could be harnessed in this way depends crucially on how 133
TREE vol. 5, no.
new variants arise. If new alleles with different numbers of repeat units are produced solely by events occurring within existing alleles, then similarities in allele maps will reflect relatedness, and historical studies should be extremely informative. If, however, there is frequent exchange or ‘crosstalk’ between alleles at meiosis, for instance by crossing over or gene conversion”, then the allele maps may be altered according to which alleles they have been in contact with. The ancestry of individual alleles will then become confused and complex, and we will need to be more circumspect about historical inferences. In an investigation of the mode and rate of change occurring within DlS8 alleles, Jeffreys et al.’ pull off their final dramatic stunt. Taking a semen and a blood sample from an individual having two similarly sized alleles, they ran the alleles out on a gel. Mutations that produce shorter alleles, with fewer repeat units, will give DNA fragments that run faster than the parental alleles. Having fine-tuned the PCR technique to the level of amplifying single molecules, Jeffreys er al. turned it loose on aliquots of the gel that might or might not have contained single DNA molecules constituting short mutant alleles, and then subjected any products to the internal mapping procedure. With this novel and quick method for assessing the frequency of new mutations (compared with searching for new variants in pedigrees), Jeffreys era/. found 64 new deletion mutants in the sperm sample and 42 in the blood sample, and calculated that deletion mutants at Dl S8 arise at a rate of about 0.007 per haploid genome. At the same time, the internal maps of the deletion mutants allow Jeffreys et al. to see the rearrangement of repeat units by which each mutation comes about.
The first revelation from this work is that all the observed mutants could be interpreted as straightforward deletion derivatives of one or other parent allele. Second, there is a mutational bias, with deletions more likely to occur at the 3’ end of the alleles. This is consistent with the finding (above) that alleles can be grouped on the basis of the maps of their 5’ ends. An important point about these results is that because no examples were found where new alleles received one end from one parent allele and the other end from the other, meiotic or mitotic crossing over between alleles seems unlikely to be a major force in the generation of new mutants. This finding, in keeping with other evidence reviewed elsewhere12,13, downgrades the hypothesis that fingerprint loci are recombination hot spots promoting crossing and interallelic over2, Jeffreys et al. now favour withinallele processes, such as unequal crossing over between sister chromatids, as the mechanism responsible for generating changes in repeat-unit number’. The fact that crossing over between alleles was not observed is also good news for the future of historical studies. However, these conclusions should, perhaps, be interpreted cautiously. Jeffreys et a/. only screened mutations that had resulted in a large reduction in allele length, which could have biased the type of mutation detected. Other processes could be at work, generating similarly sized or larger alleles than the parent alleles14. behavioural ecologists While around the world have been grappling with the vagaries of conventional DNA fingerprinting, the Leicester group have expanded the technology, substantially increasing its range of potential uses. Given the technical wizardry involved, we
5,
May 1990
predict a long gestation before the new technique is employed in nonhumans. Meanwhile, there are still many unanswered questions surrounding the generation of variability within minisatellite arrays. Why are changes in repeat-unit number in DlS.8 alleles more likely to appear at the 3’ than the 5’ end? Is the proportion of subunits with Haelll sites changing over time? How often do new restriction sites arise and move through the repeat-unit population? Is DlS8 a typical minisatellite locus or a bizarre rarity? Whatever the answers, it seems likely that Alec Jeffreys and his team will be in the vanguard providing them! References 1 Jeffreys, A.J., Neumann, R. and Wilson, V. (1990) Ce//60,473-485 2 Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Nature 314,67-73 3 Burke, T. (1989) Trends Ecol. /Sol. 4, 139-144
4 Wong, Z., Wilson, V., Jeffreys, A.J. and Thein, S.L. (1986) Nucleic Acids Res. 14, 4605-4616 5 Wong,
Z., Wilson, V., Patel, I., Povey, S. and Jeffreys, A.J. (1987) Ann. Hum. Genet.
51,269-288
6 Gill, P., Jeffreys, A.J. and Werrett, D.J. (1985)
Nature
318,577-579
7 Gill, P., Lygo, J.E., Fowler, S.J. and Werrett, D.J. (1987) Electrophoresis 8, 38-44 8 Lander, 501-505
E.S. (1989) Nature
339,
9 Cann, R.L., Stoneking, M. and Wilson, A.C. (1987) Nature 325,31-36 10 Wainscoat, J.S. et al. (1986) Nature 319,491-493 11 Dover, 159-I 65 12 Dover, 347-348
G.A.
(1986)
Trends
Genet.
G.A.
(1989)
Nature
342,
2,
13 Jarman, A.P. and Wells, !?.A. (1989) Trends Genet. 14 Dover, G.A.
5,367-371 Nature (in press)
Editor’s note This article is also appearing in Genetics.
in Trends
2 Caraco, T. (1980) Ecology 61, 119-128 3 Real, L.A. and Caraco, T. (1986) Annu. Rev.
Ecol.
Syst.
17, 371-390
4 Stephens, D.W. (1981) Anim.
Behav.
29,628-629
5 Houston, A.I. and McNamara, J.M. (1982) Anim. Behav. 30,1260-l 261 6 McNamara, J.M. (1984) SIAM J. Control
Opt/m.
2,87-94
7 Houston, A.I. and McNamara, J.M.
(1985) Behav. Ecol. Sociobiol. 17, 136-141 8 McNamara, J.M. and Houston, A.I. (1990) in Living in a Patchy Environment
5,
May 1990
11 Mazur, J.E. (1986) J. Exp. Psycho/. Anim.
Behav.
Processes
12,116-124
(Swingland, I. and Shorrocks, B., eds), pp. 23-43, Oxford University Press 9 Caraco, T., Martindale, S. and Whittam, T.S. (1980) Aim. Behav. 28,820-831 10 Herrnstein, R.J. (1964) J. fxp. Anal.
12 Staddon, J.E.R. and Reid, A.K. (1987) in Foraging Behavior (Kamil, J.R., Krebs, J.R. and Pulliam, H.R., eds), pp. 497-524, Plenum 13 Caraco, T., Blanckenhorn, W.U., Gregory, G.M., Newman, J.A., Recer, G.M. and Zwicker, S. (1990) Aim.
Behav.
Behav.
7,179-182
39,338-345
DNA Fingerprinting: ANewDimension JosephinePembertonand Bill Amos DONE IT AGAIN! Those friendly people who brought you DNA fingerprinting have now revealed genetic variation on an extraordinarily fine scale. Not only that: in a recent paper describing DNA sequence differences between alleles at a minisatellite (fingerprint) locus, Alec Jeffreys, Rita Neumann and Victoria Wilson of Leicester University, UK, have also answered fundamental questions about the rate and mode of generation of this variation’. DNA fingerprints2z3, those unevenly spaced ladders of bands, which in many species are unique for an individual, are the result of screening several extremely variable loci simultaneously. Each band represents an allele at a particular locus and is thus one of a pair. Its partner is usually elsewhere in the fingerprint profile (heterozygous state) but may occasionally occur at the same point (homozygous state). The DNA sequences detected by fingerprint probes are very short (e.g. 16 base pairs for the Jeffreys probe h33.15) and often occur in strings, known as tandem repeat arrays or minisatellite DNA. Variation in allele position on a fingerprint profile is due to differences in the length of the DNA fragments detected, which in turn result from variation in the number of repeats within an array. At minisatellite loci, repeat units appear to be easily gained or lost and different alleles can have anything from a handful to hundreds of repeats. It is this variation in repeat-unit number that causes the variation seen in DNA fingerprints. Although DNAfingerprintsare useful for determining close kin, for many other purposes (e.g. population surveys) it is useful to have locus-specific information. Unfortunately, sorting out which bands are THEY’VE
Josephine Pemberton and Bill Amos are at the Dept of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
132
allelic to one another in a DNA fingerprint is extremely difficult. The secret lies in finding conditions under which the DNA probe sticks to alleles at one locus alone, thus stripping away the confusing plethora of bands from other loci. This is achieved by first cloning, and then probing with, the DNA sequence specific to one locus, instead of using a generic minisatellite probe. The Leicester group is one of the few to have succeeded in this task. For a series of human single-locus fingerprint probes they have found locus-specific heterozygosities of 90-99% (Refs 4, 5). The latest paper’ takes this work
into a new dimension. From the outset it was clear that the repeat units within an allele, although very similar, do show subtle variations in sequence. These sequence variants presumably arise initially by point mutation, but since repeat units may share the same mutatior?, other processes must spread them between repeat units. To score such variation, one could sequence entire alleles, but the Leicester team avoided this brainnumbing exercise with an elegant ruse. Alleles at the human minisatellite locus that they investigated, called DlS8 (detected by probe MS32), have a repeat unit that is 29 base pairs long. All repeat units contain a recognition sequence for the restriction enzyme Hinfl. However, some repeat units-but not all - have
Allele A
Allele B
* 1 1L 1 1,! lw! 1 I* ! I*! 1 1
l l . l
Hid
*
*l *-
*_______--____-------------l _____-____-------------l ______----mmwm--*____---mmmmmm l _ _ _ _ -
_ -
Hinfl
H&II
Hi&
Had11
_--------__
--
------
-------_-_-
------
------
__----
Fig. 1. Schematic representation of how the internal mapping technique developed by Jeffreys eta/. can distinguish two alleles, A and 6. Top panel: Alleles are purified and radioactively labelled at one end 1”). While all repeat units in both A and B have a Hinfl restriction site (unbroken arrows), only a proportion also have a Haelll site (broken arrows). Middle panel: Partial digestion with Hinfl gives a population of end-labelled DNA fragments with one size class for each repeat unit. Partial digestion with Haelll gives fragments for only those repeat units with Haelll sites. Bottom panel: When electrophoresed and exposed to X-ray film, the Hinfl partial digests give complete ladders of bands, while the Haelll partial digest ladders have gaps. The precise position of the Haelll sites is readily determined by reference to the Hinfl ladder. Alleles A and 6, though having the same number of repeat units, are clearly distinguishable on the basis of their Haelll ladders.
TREE vol. 5, no. 5, May 7990
9
14
8
FHFHFHFH an additional recognition sequence, this time for the restriction enzyme Haelll. The beauty of the new work lies in the way in which this second source of variation is detected and read. After the polymerase chain reaction (PCR) had been used to make many exact copies of the DNA sequence of a particular allele, each molecule was radioactively labelled at one end and then aliquots were subjected to partial digestion with either Hinfl or Haelll. In a partial digest, insufficient restriction enzyme and/or time is allowed for the DNA to be cut at all recognition sites. Under the correct conditions, a population of DNA molecules is generated in which each possible cutting site is represented by end-labelled fragments of a discrete length (Fig. 1). When the products of a Hinfl partial digest are run on a gel, the result is an evenly spaced ladder of bands increasing in size by one repeat unit at a time. In contrast, the products of a Haelll partial digest result in an incomplete ladderthat lacks rungs because some repeat units are not cut by Haelll. If the two samples are run next to each other, the precise position within an allele of repeat units lacking a Haelll site can be mapped with reference to the Hinfl ladder of bands (Figs 1 and 2). Hence, each allele can be described by a binary internal map indicating the distribution of Haelll sites along it. The extra variability revealed by this technique is no less staggering than that revealed in the first DNA fingerprints. Among 32 DlS8 alleles amplified from humans of various origins and internally mapped, only two identical alleles were found. Importantly, alleles so similar in length that they are indistinguishable on a conventional fingerprint gel can have very different internal structures. Combining length variation with internal structure variability, Jeffreys eta/, calculate that for this locus alone they are now in a position to detect about 1070 different character states, should they exist! An important application of this technique will be in individual identification of criminals. Conventional DNA fingerprinting has already revolutionized forensic research6f7, but it does have an Achilles heel. For a host of reasons, not all gels run perfectly straight and it is sometimes extremely difficult to decide if two bands are identical or not. For example, in a recent double murder case in the Bronx, New York, DNA fingerprint evidence was disallowed as a result of a challenge to an interpretation that bands on a gel matched*. In future, lawyers will,
9
16 F
14 H
F
8
16
HFHFH
Fig. 2. Examples of internal mapping of four alleles (numbers 9,14,8,16) at DlS8. Each allele is represented by the gel electrophoresis pattern of a Hinfl partial digest IF) and a Haelll partial digest (H). Samples were electrophoresed on a standard gel (left) and an extension gel (above) to give maximum resolution throughout each allele. Reproduced with permission from Ref. 7.
rightly, be on the alert for such problems. The new technique not only increases accuracy by giving an exact measure of the length of an allele (from the number of repeat units detected by the Hinfl digest), but also gives a precise binary description of repeat unit types (from the Haelll digest). Furthermore, in contrast to conventional DNA fingerprints, the information is in a perfect format for storage in a computer database. It will surprise no-one to learn that the technique is the subject of patent applications. Comparing the internal maps of the 31 identified alleles, Jeffreys et al.’ observed distinct similarities
between certain alleles, including alleles of quite different lengths, especially at their 5’ ends. On the basis ofthese similarities, the authors identify four distinct groups, of which two (called 2A and 28) look closely related. This immediately raises the question of whether DlS8 alleles could be used to investigate the genetic history of the human race, shedding light on the growth and movement of peoples in the same way that mitochondrial DNA9 and other variable DNA regionslo have been applied. Whether internal mapping of fingerprint alleles could be harnessed in this way depends crucially on how 133
TREE vol. 5, no.
new variants arise. If new alleles with different numbers of repeat units are produced solely by events occurring within existing alleles, then similarities in allele maps will reflect relatedness, and historical studies should be extremely informative. If, however, there is frequent exchange or ‘crosstalk’ between alleles at meiosis, for instance by crossing over or gene conversion”, then the allele maps may be altered according to which alleles they have been in contact with. The ancestry of individual alleles will then become confused and complex, and we will need to be more circumspect about historical inferences. In an investigation of the mode and rate of change occurring within DlS8 alleles, Jeffreys et al.’ pull off their final dramatic stunt. Taking a semen and a blood sample from an individual having two similarly sized alleles, they ran the alleles out on a gel. Mutations that produce shorter alleles, with fewer repeat units, will give DNA fragments that run faster than the parental alleles. Having fine-tuned the PCR technique to the level of amplifying single molecules, Jeffreys er al. turned it loose on aliquots of the gel that might or might not have contained single DNA molecules constituting short mutant alleles, and then subjected any products to the internal mapping procedure. With this novel and quick method for assessing the frequency of new mutations (compared with searching for new variants in pedigrees), Jeffreys era/. found 64 new deletion mutants in the sperm sample and 42 in the blood sample, and calculated that deletion mutants at Dl S8 arise at a rate of about 0.007 per haploid genome. At the same time, the internal maps of the deletion mutants allow Jeffreys et al. to see the rearrangement of repeat units by which each mutation comes about.
The first revelation from this work is that all the observed mutants could be interpreted as straightforward deletion derivatives of one or other parent allele. Second, there is a mutational bias, with deletions more likely to occur at the 3’ end of the alleles. This is consistent with the finding (above) that alleles can be grouped on the basis of the maps of their 5’ ends. An important point about these results is that because no examples were found where new alleles received one end from one parent allele and the other end from the other, meiotic or mitotic crossing over between alleles seems unlikely to be a major force in the generation of new mutants. This finding, in keeping with other evidence reviewed elsewhere12,13, downgrades the hypothesis that fingerprint loci are recombination hot spots promoting crossing and interallelic over2, Jeffreys et al. now favour withinallele processes, such as unequal crossing over between sister chromatids, as the mechanism responsible for generating changes in repeat-unit number’. The fact that crossing over between alleles was not observed is also good news for the future of historical studies. However, these conclusions should, perhaps, be interpreted cautiously. Jeffreys et a/. only screened mutations that had resulted in a large reduction in allele length, which could have biased the type of mutation detected. Other processes could be at work, generating similarly sized or larger alleles than the parent alleles14. behavioural ecologists While around the world have been grappling with the vagaries of conventional DNA fingerprinting, the Leicester group have expanded the technology, substantially increasing its range of potential uses. Given the technical wizardry involved, we
5,
May 1990
predict a long gestation before the new technique is employed in nonhumans. Meanwhile, there are still many unanswered questions surrounding the generation of variability within minisatellite arrays. Why are changes in repeat-unit number in DlS.8 alleles more likely to appear at the 3’ than the 5’ end? Is the proportion of subunits with Haelll sites changing over time? How often do new restriction sites arise and move through the repeat-unit population? Is DlS8 a typical minisatellite locus or a bizarre rarity? Whatever the answers, it seems likely that Alec Jeffreys and his team will be in the vanguard providing them! References 1 Jeffreys, A.J., Neumann, R. and Wilson, V. (1990) Ce//60,473-485 2 Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Nature 314,67-73 3 Burke, T. (1989) Trends Ecol. /Sol. 4, 139-144
4 Wong, Z., Wilson, V., Jeffreys, A.J. and Thein, S.L. (1986) Nucleic Acids Res. 14, 4605-4616 5 Wong,
Z., Wilson, V., Patel, I., Povey, S. and Jeffreys, A.J. (1987) Ann. Hum. Genet.
51,269-288
6 Gill, P., Jeffreys, A.J. and Werrett, D.J. (1985)
Nature
318,577-579
7 Gill, P., Lygo, J.E., Fowler, S.J. and Werrett, D.J. (1987) Electrophoresis 8, 38-44 8 Lander, 501-505
E.S. (1989) Nature
339,
9 Cann, R.L., Stoneking, M. and Wilson, A.C. (1987) Nature 325,31-36 10 Wainscoat, J.S. et al. (1986) Nature 319,491-493 11 Dover, 159-I 65 12 Dover, 347-348
G.A.
(1986)
Trends
Genet.
G.A.
(1989)
Nature
342,
2,
13 Jarman, A.P. and Wells, !?.A. (1989) Trends Genet. 14 Dover, G.A.
5,367-371 Nature (in press)
Editor’s note This article is also appearing in Genetics.
in Trends