JOURNALOF PATHOLOGY, VOL.

REVIEW ARTICLE-CHROMOSOME

164: 101-106 (1991)

PATHOLOGY

DNA FINGERPRINTING PAUL G. DEBENHAM

Cellmark Diagnosfics, Abingdon Business Park, Abingdon, Oxon OX14 ID Y , U.K.

BACKGROUND

DNA ‘finger~rint’.~ The one exception is monozygous twins who share the same DNA fingerprint Variations in DNA sequences between individ- because they originate from the same fertilized egg uals have played a major role both in the mapping of and thus have identical DNA. In fairly rapid succession, a series of papers was human genes and in the diagnosis of inherited disorders. However, the mean heterozygosity of DNA published indicating the applicability of this new ,~ caseis low ( - 0.001 per base pair) and the chances of technology to family a n a l y ~ i s immigration detecting informative variations are very limited in- work,s and forensic investigations.6Since 1985there deed if the DNA sequences involved are not already have been ever-diversifying applications of DNA known. In 1980, a DNA sequence was discovered by fingerprinting by research scientists around the chance’ which showed hypervariability between world, resulting in tens of publications. In 1987, ICI individuals and thus it could act as a highly informa- Diagnostics* established its Cellmark Diagnostics tive marker for any nearby genes. Subsequently, a businesses at Abingdon in the U.K. and at few other hypervariable loci were discovered by Germantown, Maryland, U.S.A. to provide a comchance near the human insulin gene, the a-globin mercial DNA fingerprinting diagnostic service to genes and the c-Ha-ras- 1 oncogene. In each case, the the world, The interest has been tremendous, and in variable region consisted of side-by-side ‘tandem’ the U.K. alone, Cellmark Diagnostics has provided repeats of a short sequence (or ‘minisatellite’) in the DNA analysis reports on over 39 000 individuals to DNA, and the hypervariability of the region date in a wide range of applications. The variability of minisatellite DNA regions is between individuals was due to differences in the not constant but differs greatly. Some of the most number of repeats of the minisatellite. ~*~ In 1984, Professor Alec Jeffreys of Leicester hypervariable loci have now been i ~ o l a t e dand University’s Genetics Department discovered a used in their own right as probes for identification minisatellite region close to the human myoglobin purposes. These probes are known as single locus gene. In a study to look at the possible relatedness of probes because they analyse a single hypervariable these hypervariable regions, Professor Jeffreys iso- location in human DNA. Such loci are so variable lated this minisatellite sequence from the myoglobin that most often the copy of this DNA that we inherit gene and then used it as a probe (see ‘The Tech- from each parent differs in the number of mininique’ section) to investigate human DNA. Jeffreys satellite repeats present, thus two distinguishable discovered that the minisatellite probe result was a DNA copies can be detected. Single locus probes complex pattern of bands for each individual, some- have a major role in forensic casework because they what akin to a ‘bar-code’. In fact, the probe was have a far greater detection sensitivity than the detecting a large number of hypervariable regions in DNA fingerprint probes. The DNA fingerprint human DNA that were all related to each other, probes have become known as multi-locus probes, each band representing one of the hypervariable *Multi-locus probes 33.6 and 33.15 are the subject of patent DNA regions.‘ Further refinement of the DNA No. GBA 2166445 and worldwide patents (pending) for comsequences and techniques involved led to the recog- mercial diagnostic use. The single locus probes are also the subnition that the ‘bar-code’-like result provided a ject of worldwide patents (pending) for commercial diagnostic unique identification pattern for each individual, a use. 0022-341 7/91/050101-06 $05.00 @ 199 I by John Wiley & Sons, Ltd.

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in contrast to the single locus probes, because they detect the hypervariable regions in multiple locations in human DNA. The development of DNA fingerprinting has not precluded the original interest in hypervariable regions for medical research and such probes continue to be important in medical genetic research today. THE TECHNIQUE The technique used to study these hypervariable sequences utilizes the fact that the DNA molecule can be cut at specific recognition sites by proteins called restriction enzymes to generate millions of small DNA fragments. The lengths of the resultant DNA fragments, which contain the minisatellite regions, will change depending on how many repeats are present. DNA has to be isolated from samples provided for testing. In paternity disputes this will normally be blood samples, whereas in forensic casework the samples can range across the total spectrum of tissue and may, for example, be as small as a single hair root. After isolation, the DNA is reduced to an analytical size by treatment with a restriction enzyme and the resulting DNA fragments are then assorted by their molecular size into a long thin track in an electrophoresis gel. The track of DNA fragments produced by this step is then transferred onto the surface of a nylon membrane, where the DNA becomes permanently bound. In this bound state, the hypervariable DNA sequences in a person’s DNA will react with synthetic, laboratory-produced radioactive copies of themselves known as ‘probes’. Subsequent detection of the bound radioactive probes will thus identify the position of the hypervariable sequences in a person’s DNA. This detection is achieved by placing X-ray film against the DNA track on the nylon membrane. The radioactive probes expose the film, and thus, when developed, the film has a dark band wherever a hypervariable sequence exists in that person’s DNA. The hypervariable sequence has given a characteristic length to the DNA fragment which contains it. Thus, the position of every band in a DNA fingerprint is characteristic of an individual’s hypervariable regions. In this way, the individual’s track of DNA fragments has been converted into a simple, but unique, banded pattern ready for analysis. Single locus probes, which look at a single hypervariable region of DNA, are used in very much the

same manner as the multi-locus probe technique described above. However, in single locus probe analysis, the probes, made radioactive in the laboratory, detect just two bands in the DNA track rather than a multitude of bands. APPLICATIONS Individual identlJication We are all different from each other, as a consequence of many small differences in thousands of our genes. To distinguish each of us uniquely by tests looking at the products of these genes would therefore take many hundreds of tests. Individuality can be established to some degree by looking at 20 or more gene polymorphisms of blood cell enzymes or surface markers. Such serological tests have become well established throughout the world as a limited means of cataloguing individuality or analysing human pedigree. The advent of DNA fingerprinting has, however, revolutionized the identification ofany biological specimen by dramatically reducing the number of tests required, yet radically increasing the power ofidentification achieved. Importantly, however, the DNA sequences analysed in the DNA fingerprint are unlikely to be components of the genes that underlie characteristics such as an individual’s race, health, or intelligence. The DNA fingerprint is simply a unique pattern distinguishing an individual’s DNA, and no more than that. During his research Jeffreys isolated i! collection of different multi-locus probes and of these, two in particular, called 33.6 and 33.15, have been extensively worked with, as each provides a unique and independent DNA fingerprint pattern.’ The statistics for multi-locus probe analysis have developed from an extensive experimental basis rather than from genetic theory. In a conservative analysis, approximately one in four bands positionally match between the DNA fingerprints of two unrelated individuals.’ If two DNA fingerprints contain the same, and only the same, say 20 bands, then the odds that these two DNA fingerprints come from different persons are less than 0.2520,or less than 1 in 1 0 ‘ ~people. In a current review of paternity cases analysed by DNA fingerprinting a t Cellmark, we, in fact, identified, on average, a total of 34 bands per individual, combining the data from the probes 33.6 and 33.15. Working with good biological samples, such as the blood samples used in paternity casework,

DNA FINGERPRINTING

Fig. I-Left: A mock example of a murder case. The DNA fingerprints indicate that the specimen blood stain found on the victim is not that of the victim. There are three suspect assailants and it can be seen by comparison of the DNA fingerprints from blood samples from these suspects that the blood on suspect I matches that found on the victim. Right: An example of a paternity determination. The DNA fingerprint band pattern of the child (C) must be a composite of that from his two true parents. Thus, by comparison of the DNA bands between the child and his mother (M), it is possible to identify those DNA bands which must have been inherited from his biological father. Thus, by analysis of the DNA fingerprints of the two possible fathers (F1 and F2), only F2 can be the father of the child

allows resolution of the full DNA fingerprint information (Fig. 1). However, if a sample is degraded or limiting in quantity, then the full DNA fingerprint will not be resolved and the statistical confidence of the result will be reduced to reflect the number of DNA fingerprint bands detected. Forensic casework, a major and high-profile application of DNA fingerprinting, usually involves the identification of

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less than ideal biological samples left at the scene of a crime, such as a blood stain, semen swab, or hair roots (Fig. 1). Amongst over 1900 forensic samples analysed to date, an extremely wide range of combinations of samples, surfaces, and ages have been examined. Table I provides a broad list of samples and sources which have yielded DNA suitable for analysis to date. Importantly, the DNA fingerprints are unaffected by microbial contamination.6 In forensic casework, the term ‘DNA profile’ has become the norm, rather than the original ‘DNA fingerprint’, partly to avoid confusion with the skin pattern, but mainly because of concern that the complete DNA pattern may not be fully resolved in the limiting samples available for analysis. Thus, for forensic casework, approximately 80 per cent of the DNA tests are principally based on a more sensitive analysis of hypervariable DNA sequences using single locus probes (SLPs). The sensitivity of these probes is such that a single hair root can be identified.’ Results can also be obtained from degraded DNA, often found in forensic samples, where the SLP detects the remaining non-degraded alleles among the DNA fragments. The DNA profile obtained by use of an SLP provides just two bands to analyse, one each from the two chromosomal copies of that DNA Some SLPs have more than 100 detectable alleles and each allele is of relatively low frequency in the population. Thus, the successive application of a series of SLP tests to a forensic DNA sample can build up a near unique identification. DNA profiling evidence is now regularly submitted to courts both in the U.K. and in the U.S.A. In other parts of the world, the first cases using DNA evidence have also now been to court in The Netherlands, Germany, Sweden, and Australia, with many more countries soon to follow suit. Analysis of relationships Each person inherits half of his or her DNA from each of his or her two true biological parents, and thus a child’s DNA fingerprint is a composite of his or her parents’ DNA fingerprints. The DNA fingerprint for an individual is a sampling of information thus, it from an estimated 60 hypervariable provides extensive data from which to establish relationships. In the past, conventional serology has been able to exclude relationships with certainty but has been far less capable of establishing the converse, i.e. the presence of a relationship beyond doubt; DNA fingerprinting now provides this long sought after definition.

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Table I-Casework samples Blood

Semen

-EDTA bIood/heparin blood -Clotted blood --Stains on -Cloth -Wood -Metal -Plastic -Floor tiles -Wallpaper/newspaper -Food

--Stains on cloth, on paper -On vaginal, anal, and oral swabs -Vaginal aspirates -MOSS -Fur

DNA analysis of disputed relationships such as alleged paternity is at its most powerful when both parents are available for testing along with the child. The samples involved are then processed together and the DNA fingerprints compared. By comparison of the child’s DNA fingerprint with that of the mother, all possible maternally inherited DNA fingerprint bands can be identified. The remaining bands in the child’s DNA fingerprint must be inherited from the true biological father. In a current review of paternity cases resolved by DNA fingerprinting, the child’s DNA fingerprints (33.6 and 33.15) contained, on average, a total of 15 nonmaternal bands. The chance match of 15 such bands can be calculated, as for identity analysis, as 0.25”, that is, 1 in 1 000 000 000 individuals. Thus, positive identification of paternity, or maternity, can be established. Whilst the present statistical approach to paternity analysis has not been faulted in the thousands of cases now reported, new biostatistical approaches have now been developed which take into account the distribution of all the DNA fin er print bands, not just those of paternal origin.” 1; the vast majority of cases, such alternative approaches are not likely to greatly influence the significance ofthe results obtained, and may, in fact, provide greater weight to analyses involving low numbers of paternal specific bands. The natural processes of genetic change which, through evolution, have generated the diversity recorded in our present DNA fingerprints, are, of course, still occurring. New ‘mutant’ hypervariable regions, differing from

Hair

-Head, body, and pubic hair -Hair and scalp samples

Tissue

-Bone marrow -Muscle --Spleen -Fingernail scrapings -Leukaemic cells

Mouth

-Swabs

Fetus

-Muscle biopsy -Chorionic villus sample

those of the parents, are thus to be expected and probably occur by a process of unequal cross-over between alleles on sister chromatids or by replication slippage.”,2’ The occurrence of such a band in a paternity analysis thus requires consideration of various parentage permutations as alternative explanations for the unmatched band. However, in the context of the large number of informative DNA fingerprint bands, such hypotheses are normally of minimal significance.12 DNA fingerprinting can be informative, though not necessarily conclusive, in cases where only one parent is alive. It is possible to reconstruct a missing parent’s DNA fingerprint from his ~ h i l d r e n , ~ though often this cannot be completely achieved. Alternatively, an analysis of the similarity of the DNA fingerprints of two allegedly related individuals can be compared and the degree of similarity can indicate the most probable relationship involved. A combination of results from both multilocus and single locus probes can together further confirm relationships where a full pedigree is not available. Sometimes, alternative tissue samples can be used in place of the missing person. Post-mortem samples have been utilized in some cases of testacy dispute, and fetal samples from abortions have been informative in cases of under-age sex or rape where paternity is to be determined. In the U.K., DNA fingerprinting results are now fully accepted by the Home Office as proof of relationships in immigration a peal cases, following a report published in 1988.’- In 1989, the Home

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Office gave official recognition to the role of DNA are a true hybrid of both cell types, and that the fingerprinting in paternity disputes by granting Cell- hybrid status is maintained during extended culture. mark Diagnostics official Registered Tester status DNA fingerprinting, of course, can also play a key role in monitoring ordinary cell lines in the under the Blood Tests (Evidence of PaternityAmendment) Regulations 1989. The Home Office is laboratory. now actively considering the establishment of a centrally organized scheme for disputed relationships in THE FUTURE immigration casework which will almost certainly ICI Diagnostics, through its Cellmark Diaginvolve DNA fingerprinting at the time of application rather than after years of appeal procedures. nostics businesses, is now actively engaged in Thus, DNA fingerprinting has become the key spreading the technology around the world. Sublicensing agreements are being established in many determinant in issues of relationship. Even the Kennel Club has recognized DNA countries whereby private laboratories and forensic fingerprinting in the resolution ofdisputed paternity laboratories can provide the benefits of the techindogs! Themulti-locusprobes33.6and33.15detect nology to their country. Thus, it can be expected to DNA fingerprints not only in humans’ DNA, but be a universally applied technology in the foreseein birds,I6 and, in fact, all vertebrates able future. Single locus probes represent the first analysed to date, although some are not as informa- major development of the technology since its tive as those obtained for humans. The single locus original discovery and further developments are expected in the not-too-distant future. The techprobes are, however, human-specific. nology itself has already gained wide acceptance Laboratory applications and usage in several countries around the world, In the hospital laboratory, DNA fingerprinting and undoubtedly the future for DNA probe techcan be useful in monitoring the remission of bone nology and its application to identity analysis is marrow transplants following total body irradi- destined to be one of rapid development and even ation to destroy leukaemia, because the trans- further diversification. planted bone marrow and its blood cells will have a different DNA fingerprint to that of the tissue of the REFERENCES recipient. If the recipient’s DNA fingerprint begins I. Wyman A, White R. A highly polymorphic locus in human DNA. to be found in his or her blood, then this means that Proc Nuti Acud Sci U S A 1980; 77: 67546758. some cancerous cells could have survived in the 2. Jeffreys AJ, Wilson V, Thein SL. Hypervariable ‘minisatellite’ regions in human DNA. Nurure 1985; 314: 67-73. bone marrow and doctors will be able to take early AJ, Wilson V, Thein SL. Individual specific ‘fingerprints’ of action to eradicate them.I7 Recently, the possibility 3. Jeffreys human DNA. Nulure 1985;316: 7 6 7 9 . has been raised of using DNA fingerprinting to 4. Jeffreys AJ, Wilson V, Thein SL, Weatherall DJ, Ponder BAJ. DNA fingerprints and segregation analysis of multiple markers in human establish the relatedness of organ donors and recipipedigrees. A m J Hum Grner 1986; 3 9 11-24. ents in instances where such relationships are in 5. Jeffreys AJ, Brookfield JFY, Semeonoff. Positive identification of an immigration test-case using human D N A fingerprints. Narure 1985; doubt. In hospital research, it is often important to 818-819. define or confirm the zygosity of twins and, of 6. 317: Gill P, Jeffreys AJ, Werrect DJ. Forensic application of DNA fingercourse, DNA fingerprinting can rapidly and simply prints. Nuture 1985; 318 577-579. 7. Wong Z, Wilson V. Jeffreys AJ, Thein SL. Cloning a selected fragment address this question.’* from a human DNA fingerprint. Isolation of an extremely polymorThe DNA fingerprinting probes, whilst human in phic minisatellite. Nuclric Acid Res 1986; 1 4 46054615. origin, also detect equivalent hypervariable regions 8. Wong Z, Wilson V, Patel I, Povey S, Jeffreys AJ. ChardcteriSdtiOn of a panel of highly variable minisatellites cloned from human DNA. Ann in animals; thus, animal pedigree can be checked Hum Gener 1987; 51: 269-288. and monitored in laboratory animal stocks. For 9. Hopkin B, Morton JEN, Smith JC, Markham AF. The development instance, DNA fingerprinting can be used to analyse of methods for the analysis of DNA extracted from forensic samples. Technique 1989; 1:96-102. the degree of genetic similarity in in-bred mouse 10. Evett IW, Werrett DJ, Buckleton JS. Paternity calculations from s!rains”. In the biotechnology laboratory, DNA DNA multilocus profiles. J Forensic Sci Soc 1989; 2 9 249-254. fingerprinting can be used to authenticate tissue 1 1 . Jeffreys AJ, Royle NJ, Wilson V, Wong Z. Spontaneous mutation rates to human length alleles at tandem repetitive hypervariable loci in culture cell lines by establishing reference DNA human DNA. Narure 1988; 332: 278-281. patterns for each cell line, thus allowing clear identi- 12. Sheridan R, Llerena J Jr, Matkins S , Debenham P, Cawood A, Bobrow M. Fertility in a male with trisomy 21. JMedGener 1989; 2 6 fication of cross-contamination.20Cell hybrids can also be characterized by DNA fingerprint compari- 13. 294298. Home Office, London. DNA profiling in immigration casework. sons with the parent cell lines to establish that they London: HMSO, 1988.

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14. Jeffreys AJ, Morton DB. D N A fingerprints of dogs and cats. h i m Gene/ 1987; 1 8 1-15. 15. Morton DB, Yaxley R E , Patel I, Jeffreys AJ, Howes SJ, Debenham PG. The use of D N A fingerprint analysis in identificdlion of the sire. Vrr Rrc 1987; 121: 592-593. 16. Burke T, Bruford M W . D N A fingerprinting in birds. Narure 1987; 327: 149-152. 17. Min GL, Hibbin J, Arthur C, Apperley J , Jeffreys A, Goldman J . Use of minisatellite D N A probes for recognition and characterization of relapse after allogenic bone marrow transplantation. Br J Harmaroi 1988; 68: 195-201. 18. Hill AVS, Jeffreys AJ. Use of minisatellite DNA probes for determination of twin zygosity at birth Lrmcet 1985; ii: I 3 9 6 1 395.

19. Jeffreys AJ, Wilson V, Kelly R, Taylor BA, Bulfield G. Mouse D N A fingerprints: analysis of chromosome localization and germ-line stdbility of hypervariable loci in recombinant inbred strains. Nucleic Acid Rrs 1987; 15: 2823-2836. 20. Thacker J, Webb MBT, Debenham PG. Fingerprinting cell lines: use of human hypervariable D N A probes to characterize mammalian cell cultures. Somu/ Ce// MolGener 1988; 1 4 519-525. 21. Jeffreys AJ, Neumann R, Wilson V. Repeat unit sequence variation in minisatellites: A novel source of D N A polymorphism for studying variation and mutation by single molecule analysis. Cell 1990; 60: 473485.