Trends in Molecular Medicine

Identification of Individuals with DNA Testing Antti Sajantila' and Bruce Budowle2

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The fast technical development in molecular biology and the increasing knowledgeof the human genome has had a major impact on forensic medicine. Genetic characterization of individuals at the DNA level enables identity testing from a minimal amount of biological specimen in cases of sexual assault, homicide, and unknown human remains. Also paternity testing is changing from level of gene products to the genomic level. This article addresses some of the advantages of DNA testing over the conventionalforensic serology, and the DNA techniques currently used in forensic science. Key words: identity testing; DNA; forensic medicine. (Annals of Medicine 23: 6 3 7 4 4 2 , 1991)

Polymorphisms in the Human Genome

Table 1. Characteristics of a useful forensic genetic marker. Marker

It has been estimated that human genome exhibits polymorphismrangingfrom 1 in every 100to 1 in every 1000 nucleotides (1). Therefore, variation among humans at the DNA level is abundant for identificationpurposes. Typically, polymorphisms have been detected at the level of gene products, but variation is greater at the DNA level. Polymorphisms at the DNA level can be caused by single nucleotidevariations. At the same locus there can also exist multiple single nucleotidevariations. This kind of variability is termed as a sequence polymorphism. However, most of the variation in the human genome is found in the DNA regions that at present do not have any known coding or regulatoryfunction. In 1980Wyman and White (2) observed that genomic DNA contains multiple repeats of certain nucleotidesequences within the non-coding regions. Moreover, the number of these repeats showed to be highly variable inthe human populations resultingin polymorphisms that can be called length polymorphisms (also known as variable number of tandem repeats, VNTR). In the human genome there are also other kind of length polymorphisms due to insertions or deletions (like FA508). To be useful for identification of individuals, the markers used (whether protein or DNA markers) should fulfil certain criteria. These include knowledge of inheritance pattern from generation to generation, a high degree of polymorphism, independent inheritance from the other markers used, established allele and pheno/genotypefrequencies, and ease of analysis (Table 1). The same markers and techniques that are useful for identification of individuals are also well applicable to paternity testing (Table 2).

Table 2. Potential polymorphic markers for DNA identity testing.

From the 'Laboratory of Molecular Genetics, National Public Health Institute, Helsinki, Finland,2ForensicScience Research and Training Center, FBI Academy, Quantico, Virginia, U S A . Address and reprint requests: Antti Sajantila, M.D., Laboratory of Molecular Genetics, National Public Health Institute, Mannerheimintie 166. SF-00300 Helsinki, Finland.

VNTR = variable number of tandem repeats 'STR = short tandem repeat 3AS0= allele specific oligonucleotide 40LA = oligo ligation assay VntDNA = mitochondria1 DNA Current use in forensic laboratories

1. 2. 3. 4. 5.

Pattern of inheritance well established Polymorphic with high degree of heterozygosity Inherited independently of the other markers used Mutation rate very low Population data of allele, phenotype and/or genotype fre quency established 6. Technique used for its analysis is simple, rapid, reproducible and of reasonable cost 7. Reliable allele detection 8. Little material needed

Analysis method

Forensic use'

1. Lentgh polymorphisms - VNTR'-loci - STR2-loci

Electrophoresis Electrophoresis

Case work Case work

2. Sequence polymorphisms - single nucleotide variations

ow4

ASO-hybridization

Research Research Research

Marker

Solid-phase minisequencing

- multiple nucleotide variations

- HLA-DQa - mtDNA5

AS03-hybridization Case-work ASO-hybridization Research Research direct seauencina

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Principles in Identity Testing In identity testing the scientist actually is performing profile comparisons, i.e are the evidence sample profiles (whether DNA or protein) similar or dissimilar to samples obtained from avictim or suspect. Analysis of agiven forensic sample has three interpretations of comparisons between known and unknown sample: (i) inclusion, i.e. the questioned sample potentially originates from the same source as the reference sample, (ii) exclusion, i.e. the questioned sample and the reference sample can not be derived from same biological source, and (iii) inconclusive, where insufficient data is available concerningsamples in question. The result of a paternity test is usually expressed in the form of likelihoodratio, called the paternityindex (3). This is derived from genetic data of the mother, the child and the man in question, and from the frequencies of occurrence of particular allele of the genetic marker in the relevant population (4). When the DNA profile obtainedfrom a forensic specimen matches to that obtained from a suspect, the weight of the evidence relies on the distribution of the genotypes (or alleles) in the population in question. The genotypefrequency data in the appropriate population is essential for calculating the probability that the same DNA profile could be derived from a random, unrelated person. One issue in population genetics connected to genetic evidence is the assumptionof Hardy-Weinberg(H-W) equilibrium (56).A H-W population is one that meetsthe criteria of (i)no selection, (ii) no immigration, (iii) no emigration, (iv) no mutation, and (v) large population. It matters little whether of not a particular population meets these criteria, but it is important that the locus meets H-W expectations, i.e. can the allelic frequencies be used to predict the genotype frequencies. Essentiallythis means that if a locus meets HW expectations the alleles associate randomly. It is important to address the concept of H-W expectation when analyzing the highly polymorphic loci. For example, if there are 20 alleles at a particular locus in a particular population there can be as many as 210 genotypes. With that many possibilities it is unlikely that all possible genotypes will be observedeven with substantially large populationsamples. The assumption of H-W equilibrium permits the estimation of the frequency of occurrence of a particular genotype that has not been observed in the population previously.

Conventional Forensic Serology Since discovery of the ABO blood group system in the beginning of this century characterizationof individuals by using genetic markers has been applied to demonstrate associations or exclusions between forensic specimen and criminal suspects (or victims), and to determine familial relationships. Genetic markers in human blood (and body fluids) used in classical forensic serology were protein products and were classified generally as: erythrocyte antigens, serum proteins, erythrocyte enzymes, and the HLA-system. Analysis of erythrocyte antigens, serum proteins, and erythrocyte enzymes has been found to be informativeparticularly in paternity testing. Using these systems mentioned above 90-95 % of falsely accused men can be excluded. Whereas, by using HLA-typing alone the mean probability of exclusion is approximately the same, and by combining

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HLA-typing to erythrocyte antigens, serum proteins and erythrocyte enzymes the figure is around 98 %. In forensic analysis the use of conventional serology has been limited. The unstable nature of gene products, insufficient amount of specimen, and contaminationor mixing of samples (i.e. semen and vaginal secretions) are not easily overcome by conventional serologic methods. In practice, usually only blood group ABO, serum groups Hp, Gc, Tf (habtoglobin, group specific component, transferrin) and erythrocyteenzymes Glo, EsD, ADA, PepA, AcP, AK, PGM (glutamateoxygenase, esterase D, adenosine deaminase, peptidase A, acid phosphatase, adenylate kinase, phosphoglycomutase,) can be typed when the blood sample is dry. However, in practise not all were obtained. Vast majority of forensic cases are sexual assaults. In cases of sexual assault the situation is even less informative potentiallyonly ABO blood group and PGM can be obtained from seminal fluid. Thus, characterization of an individual from semen routinely provided little information.

Forensic DNA Analysis DNA level identity testing has revolutionizedforensic biology becauseof its powerfulpotentialof discriminationand the possibilityto analyze minuteamounts of virtually any biological specimen. Due to the remarkable methodological developments in molecular biology and increasing knowledge of the human genome DNA typing has become in reality in the 1980’s. DNA regions reveal greater variation than loci analyzed in conventionaltestintg. Therefore, better discrimination between individuals per analysis is achieved.

Methodological Development Forensic laboratories currently make use of two different methods of DNA typing: (i) hybridization analysis using either multilocus probes (MLPs) or single-locus probes (SLPs) to detect restrictionfragment length polymorphisms (RFLPs) (7),and (ii) polymerasechain reaction (PCR) (8,9) to detect either sequence polymorphisms or length polymorphisms. RFLP is a basic technique in molecular biology, which was originally developed for the detection of single base variations in the humangenome (7).The technique is based on the recognition that the human genome is replete with INDIVIDUAL VARIATION IN CLEAVAGE SITE

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.

PROBE0 REGION

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Figure 1. A schematic presentation of a RFLP caused by a point mutation.The different alleles vatying in lengthcan be separated in electrophoresis. (Adopted from Ref. 4).

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Identification of Individuals Based on DNA Testing single nucleotide variations which can be detected by the action of restrictionenzymes (Fig. 1).With a suitable restriction enzyme the DNA can be cleaved at a polymorphic site if it has a particular variant and not if it does not have it, resulting in length polymorphism. These length polymorphisms can be analyzed by using agarose gel electrophoresis and Southern blotting (10) (Fig. 2). The PCR is an enzymatic process in which a specific region of DNA is amplified in vitro to yield several million copies of a particularsequence. DNA amplificationinvolves repeated temperature cycling with three stages per cycle. The different stages are: (i) denaturationof DNA sample so that each strand can serve as template of DNA (ii) annealing of specific primers to act as starting points for copying the DNA strand, and (iii) extensionof the primerto make a copy of the target DNA strand (Fig. 3). The extension step is performed by using thermoresistantenzymeTaq polymerase derived from bacteria Thermus aquaticus. After the amplification process the PCR products can be analyzed by various methods for the detection of both sequence and length polymorphisms.

RFLP-Hybridization Analysis Using Multilocus Probes The practicalrealityof DNA identitytesting emerged in 1985 with Alec J. Jeffreys’spublicationsconcerning hypervariable “minisatellite” regions in the human genome (1 1). These regions could be analyzed using MLPs detecting several VNTR loci simultaneously. The patterns revealed after Southern blotting and MLP hybridizationwere termed individual-specific “DNA fingerprints” (12). The mean probabilitythat two randomly chosen, unrelated individuals possessed the same pattern in DNA fingerprinting analysis was calculated to be 3 x lo-” for one multilocus probe (12). Although DNA fingerprinting raised hope to achieve an absolute identification,and providedthe gratest individual specificity per analysis when compared with other DNA typing methods, the DNA fingerprinting technique had some drawbacks which decreased the desire for use in forensic case work. First, a sufficient amount of DNA is needed (at least 100 ng to 1000 ng). Second, the quality of DNA is of significance. DNA can and will degrade when exposed to the environment: this will result in loss of high molecular fragments in DNA fingerprints of forensic samples, and concomitant loss of information. Third, interpretation of the complex DNA fingerprint pattern can be difficult if two or more individualscontribute to the patterns, and because the statistical calculationsapplied to the analysis differ from that used to conventionalforensic serology. In addition, MLPs hybridize to non-human sources, for example such as yeast. However, recently, Jeff reys and the co-workers published a case work study involving 1702 paternity cases, describing the effectiveness of multilocus probes in paternity testing (13).

RFLP-HybridizationAnalysis Using Single-Locus Probes A means to overcome the limitationsof multilocusapproach for forensic DNA analysis is the use of a panel of SLPs. By using a suitable set of SLPs via Southern blotting and hybridization, it is possibleto obtain a DNA profile which can be unique to an individual. The SLPs applied to forensic or paternity testing detect alleles at specific, single loci. The

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Figure 2. A schematic presentationof Southern blotting method for detection of RFLPs.

most informative loci so far are variable number of tandem repeat (VNTR) loci (14) presenting a high degree of heterozygosity and a variety of alleles differing from each other in the length in every population tested. The length polymorphism is due to the variationof anumber of tandemly repeated core sequences. The advantages of SLP typing compared with MLP typing for forensics are (i) the amount of DNA needed is approximatelya magnitude less (10-1 00 ng), (ii) the patterns are less complex, (iii)no species crossreact with probesused (excepthigher primates).A limitation of VNTR typing via RFLP analysis is that the resolution of agarose gel electrophoresis is not optimal. To avoid the technical drawbacks some laboratories have developed protocols for the DNA analysis of forensic specimen, and computer-assisted analysis systems to assist in the interpretation of the results (15).

Polymerase Chain Reaction in Forensic Analyses The advantages of PCR compared with RFLP analysis in forensic science are remarkable. Firstly, the ability to type Ann Med 23

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Figure 3. Principles of the polymerase chain reaction.

degraded DNA enables analysis of biological specimen even in cases of old, decomposedsamples, where DNAcan be highlydegraded. Secondly, with suitable detectionmethods the measesurementerror associatedwith RFLP can be greatly reduced.Thirdly, sensitivityof PCR is overwhelming

when compared to any other method used to identify DNA sequences in biological specimens; this quality facilitates analysis of minute amounts of DNA. Fourthly, analysis time can be reducedfrom up to 6 weeks to only a couple of days. Finally, forensic DNA analysis using PCR is economic and subject to automation. The post PCR analysis strategies applied to forensic testing include analysis of VNTRsor a subset of VNTRs with a repeat length of 2-4 bp (also called short tandem repeats, STRs) by usingthe amplifiedfragment length polymorphism (Amp-FLP)technique (16) (Fig. 4). The Amp-FLPtechnique utilizes the advantages of PCR and a high resolution polyacrylamide gel electrophoresis system to detect the high degree of polymorphism and heterozygosity of VNTR and STR loci. The different alleles at VNTR or STR loci are separated by high resolution polyacrylamide gel electrophoresis, and subsequently visualized with a sensitive silver staining. In addition to the benefits of sensitivity, specificity and rapid analysis, the results obtained can be compared readily with different laboratories weather or not they used a standard analytical method. However, it has been proposed that sharing a common allelic standard would be desirable (17). The Amp-FLP technique has already been used in forensic case work and paternity testing in Finlandsincethebeginningofthisyear(l991)(18, 19). At the moment three hypervariable VNTR loci (D1S80, D17S30,ApoB) are used, but more VNTWSTR markersare under investigation. Another PCR-based approach is the analysis of sequence variation. Sequence variation can be detected by allele-specific oligonucleotide (ASO) hybiridzation (Fig. 5; 20). An example of ASO-hybridization approach is the analysis of the second exon of the highlyvariable HLA-DQa locus. This can be accomplished easily with a simple, commercially availableAmpliTypeTM HLA-DQForensicDNA Amplification and Typing Kit (Cetus Corporation, California, USA). The technique has been evaluated by the FBI laboratory for forensic use (21) and has already been applied for case work both in Finland and in the United States. In our experience, PCR has been advantageous for the investigationof sexual assaults. Also blood stain analyses

Figure 4. An example of an Amp-FLP analysis of D1S80 locus in forensic case-work. (Adoptedfrom Ref. 18). St = allele standard, S = suspect, B = bloodstain,V =victim, MW = molecular weight marker.

Identification of Individuals Based on DNA Testing

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STREPTAVIDIN HRP ENZYME CONJUGATE AMPLIFIED DNA IMMOBILIZED A S 0 PROBE

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‘A1 I el e-S pecific Oligo nucleot ide Figure 5. A schematic presentation of ASO-hybridization analysis of HLA-DQa locus using reverse dot blot technique. (Adopted from Ref. 25). DNA is amplified by PCR using biotinylated primers. The amplified product is subsequently hybridized to a filter strip carrying immobilized A S 0 probes, and the detection is enzymatically mediated by a streptavidin-horseradish peroxidase (HRP) conjugate resulting in a visually detectable dye.

and identificationof unidentified human remains has benefited from the development of DNA analysis. By using the three VNTR loci in paternity testing, the mean probability of exlusion is approximately the same as that obtained with conventional serological testing. With one or two more hypervariable markers the mean probability of exclusion of over 99 % will be achieved.

analysis of length polymorphisms in the human genome. Perhaps direct sequencing of PCR products may be the ultimate identity test in hands of forensic case work experts in future once technical limitations are obviated.

References 1. Thomas Caskey C, Edwards A, Harnmond HA. DNA: the

Future Prospects Research concerning new techniques for individual identification is active. Screening of a panel of common single base variations in the human genome could be easily automated and potentiate a high discrimination between individuals. Detectionof single base variations can be done by ASO-hybiridzation, oligonucleotideligation assay (22) or solid phase mini-sequencing(23). On the other hand, capillary electrophoresis and high performance liquid chromoatography could provide sensitive means for the

2. 3.

4. 5. 6.

history and the future use in forensic analysis. Proceedings of the international symposium on the forensic aspects of DNA analysis. June 19-23, 1989, FBI Academy, Quantico, Virginia, U S . Washington D.C. US. Government Printing Office. Wyman AR, White R. A highly polymorphic locus in human DNA. Proc Natl Acad Sci USA 1980; 77: 6754-8. Aho K, Leino U. Paternity and blood group evidence: Scandinavian practice. The International and Comparative Law Quaterly 1982; 31: 576-81. Silver H. Paternity testing. Critical reviews in clinical laboratory sciences 1989; 27: 391408.0001. Hardy GH. Mendelian proportions in mixed populations. Science 1908; 28: 49-50. Weinberg W. Uber den nachweis der vererbung beim

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9. 10. 11. 12. 13.

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14. 15.

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menschen.JahreshefteVer. Vaterland. Naturkd.Wurttemburg, Stuttgarll908; 64: 368-82. Botstein D, White R, Skolnick M, Davis R. Constructionof genetic linkage maps using restriction fragment length polymorphism. Am J Hum Genet 1980; 32: 314-31. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzedchain reaction. In: Wu R, Grossman, Moldave K, eds. Methods in Enzymology. New York Academic Press, 1987; 155: 335-50. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of P-globingenomic sequences and restrictionsite analysis for diagnosis of sicle cell anemia. Science 1985; 230: 1350-4. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-1 7. JeffreysAJ, Wilson V, Thein SL. Hypervariable “minisatellite regions in human DNA”. Nature 1985; 314: 67-73. Jeffreys,Wilson V, Thein SL. Individual-specific‘fingerprints’ in human DNA. Nature 1985; 316: 76-9. Jeffreys AJ, Turner M, Depenham P. The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships,determinedfrom extensive casework. Am J Hum Genet 1991; 48: 824-40. Nakamura Y, LeppertM, OConnell P, et al. Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 1987; 235: 1616-22. Budowle B, Giusti AM, Waye JS. et al. Fixed-binanalysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons. Am J Hum Genet 1991; 48: 841-55.

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16. BudowieB, ChakrabortyR,Giusti AM, EisenbergAJ,Allen R. Analysis of the VNTR locus D1S80 by the PCR followed by high resolution PAGE. Am J Hum Genet 1991; 48: 1 3 7 4 4 . 17. Sajantila A, PuomilahtiS, Johnsson C, Ehnholm C. Amplification of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis. Biotechniques, in press. 18. Sajantila A, Budowie B, Strom M, et al. Amplification of alleles at the D1S80 locus by the polymerase chain reaction: comparison of a Finnish and a North American population sample, and forensic case-work evaluation, submitted. 19. Helminen P, Sajantila A, Johnsson V, Lukka M, Ehnholm C, Peltonen L. Amplification (PCR) of three hypervariable regions in paternity determinations Molecular and Cellular Probes, in press. 20. Erlich HA, Bugawan TL. HLA class I I gene polymorphism: DNA typing, evolution,and relationshipto disease susceptibility. In: Erlich HA, ed. PCR technology - principles and applicationsfor DNA amplification, New York: Stockton Press, 1989: 193-208. 21. Comey CT, Budowle B. Validation studies of the analysis of the HLA-DQa locus using the polymerase chain reaction. J Forensic Sci, in press. 22. Nickerson DA. Kaiser R, Lappin S, Stewart J, Hood L, Landegren U. Automated DNA diagnostics using an ELISAbased oligonucleotideligation assay. Proc Natl Acad Sci USA 1990; 87: 8923-7. 23. SyvanenAC, Aalto-SetslaK, HarjuL, Kontula K,Soderlund H. A primer-guided nucleotide incorporation assay in the genotyping of apolipoproteinE. Genomics 1990; 8: 684-92.