J7 Med Genet 1993; 30: 625-633

REVIEW ARTICLE

Forensic medicine and the polymerase chainreaction technique

Ronny Decorte, Jean-Jacques Cassiman

DNA technology in forensic medicineForensic scientists have for decades been usingserological and electrophoretic methods to de-termine the origin of blood stains or body fluidsamples. ' The discrimination potential of thesesystems is low compared to-certain DNA poly-morphisms. Moreover, they rely on theexpression of polymorphic proteins and thepossibility of detecting a change in a physicalproperty of a protein.2 Genetic marker typingat the DNA level is, however, not restricted togenetic variation in coding sequences. In fact,the most polymorphic sites in the humangenome lie outside amino acid coding regions.Because of the higher information potential atthe DNA level and the presence of DNA inevery nucleated cell, forensic scientists intro-duced recombinant DNA technology in casework samples in the mid 1980s. At that time,Alec Jeffreys showed that highly polymorphicDNA segments (minisatellites or variablenumber of tandem repeats) are able to generateindividual specific DNA 'fingerprints' afterhybridisation under low stringency con-ditions.34 It was further shown that reproduc-ible genetic profiles could be produced fromdried blood stains (up to four years old), semenstains, hair roots, and blood stains exposed todifferent environmental conditions.9

Perhaps the greatest contribution of DNAtechnology to forensic medicine has been inthe identification of rapists. Indeed, the major-ity of the forensic evidence samples which aresubmitted for DNA analysis to our forensiclaboratory have been derived from sexual as-sault cases (table 1). In these cases, the vaginalswabs or lavages taken from the victim areoften a mixture of spermatozoa and cellulardebris (table 2). This poses a problem whenserological methods are used for the identifica-tion of different polymorphic proteins, es-

Table I Distribution according to the offence of 178case work samples submitted for examination.

Centre for HumanGenetics, University ofLeuven, CampusGasthuisberg O&N6,Herestraat 49, B-3000Leuven, Belgium.R DecorteJ-J Cassiman

Correspondence toProfessor Cassiman.

Violent offencesRape 41 7Murder 28 6Homicide 9 0Robbery 7-3Assault 1-7Kidnapping 1.1

Other offencesCar accidents 1 1Burglary 1 1Blackmail 1 1Others 7-3

pecially when the number of spermatozoa arequite low. However, it is possible to separateDNA of the sperm nuclei from DNA of theepithelial cells.67 The protein coat of the spermhead contains disulphide bridges that can bebroken by dithiotreitol (DTT). By differentiallysis with a detergent and a proteinase, DNAof the contaminating cells is released while thesperm heads will remain intact (figs 1 and 2B).The semen cells are recovered by centrifugationand subsequently lysed in the presence ofDTT.

Initial procedures for detecting highly poly-morphic loci relied on Southern blots andradioactively labelled DNA probes. Thesemethods are sufficiently sensitive (up to 60 ng)to allow the detection of the different alleles inDNA samples of high molecular weight'0: thisis usually the case for DNA extracted fromblood samples in paternity cases. However,forensic samples in criminal cases do notalways contain sufficient non-degraded DNAfor analysis of variable number of tandemrepeats (VNTR) loci. A report on the stabilityof DNA in actual case work showed that in48% of the samples either degraded or unde-tectable DNA was present."I Another potentialproblem is the low resolution of the agarosegels used for sizing restriction fragments. Thesize differences between two alleles can bebetween 9 and 70 base pairs and large DNAfragments with such a difference will migrateclose to each other and could be typed as oneallele instead of tWo.'213

Forensic DNA typing and PCRThe problems associated with DNA typing offorensic biological samples (for example, in-sufficient DNA, degraded DNA, resolution ofthe gels) can be resolved in part by the poly-merase chain reaction technique. PCR basedtyping is simple and has a high sensitivity.'4Moreover, it decreases the analysis time to lessthan one day compared to conventional South-ern blot analyses which require at least one

Table 2 Biological evidence samples received forexamination.

Mixed samples: semen+ blood or epithelial cells 41 4hair 6-8

Stains of blood or semen (on clothing, glass, knives, etc) 40 9Hair 3-2Saliva (cigarette butts, envelopes, stamps, etc) 2-8Others (bones, teeth, body pieces, etc) 4-9

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-0 rI~~ ~

-.-0o

03Detergent

DDTDetergent

t¢) Proteinase KDNA

Figure 1 Differential lysis procedure for separation of a mixture of spermatozoa fromithe assailant and epithelial cells from the victim.

week. Degraded DNA is suitable as a substratefor amplification as long as there is at least oneDNA fragment present encompassing the two

primer binding sites.'3 The primer sites can bedesigned to flank directly the repeat regions inVNTR loci so that small differences of one

repeat unit can be detected after size fractiona-tion on high resolution gels. This minimisesthe problem of incomplete resolution of allelesand as a consequence leads to more correctallele frequency estimates.'3 1" In addition, it is

possible to construct for each VNTR locus a

reference size marker composed of the dif-ferent amplified alleles observed in a popula-tion. This marker is then simultaneously sizefractionated with the PCR products to beanalysed (fig 2B). It allows for rapid and more

correct interpretation of the amplified frag-ments.'7 Moreover, a nomenclature based on

the number of repeats can be used to label thedifferent VNTR alleles. This would improve

.40 so

Figure 2 Detection of amplified VNTR loci by different methods. (A) Segregation of the IGHJ VNTR alleles in

a three generation family, size fractionated in a 2% agarose gel, and visualised with ethidium-bromide ( + = positivecontrol, - = negative control, m = 123 bp ladder (BRL) ). (B) Analysis with the VNTR Dl 7S5 of two bloodsamples (victim and suspect) and two semen stains (I and 2) on the clothing of the victim, extracted by differentiallysis (A = contaminating DNA from the victim, B = DNA from semen, m = reference marker of amplified alleles).Amplified fragments were labelled during PCR with biotin-1J-dUTP and detected after Southern transfer withavidin alkaline phosphatase and chemiluminescence. The result showed no contaminating cells from the victim.(C) Population screening with the APOB VNTR: alleles were amplified with one of the primers labelled withfluorescein; PCR fragments were size fractionated in a 4% Hydrolink Long Ranger gel on a Pharmacia ALFautomated DNA sequencer.

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Forensic medicine and the polymerase chain reaction technique

standardisation and allow for exchange of databetween forensic laboratories.

Because of the high sensitivity of PCR, onedoes not need large amounts of DNA fortyping polymorphisms. This has the advantagethat repeated analysis is possible in case oftechnical failure or when confirmation of theresults is necessary. Defence lawyers arebecoming aware of the high inclusion probabi-lities with DNA typing methods and are oftenrequesting a second opinion.'8 '9 Therefore, ifonly minute DNA samples are used for theanalysis, enough DNA will be left for a secondanalysis in another forensic laboratory. In con-trast, conventional DNA typing needs at least50 to 500 ng of high molecular weight DNA,and in most cases repeat analysis is not pos-sible.The use of PCR for the analysis of DNA

from evidence samples has also made it pos-sible to use other material which could connectcertain subjects with a crime (table 2). Refer-ence DNA samples from suspects and victimsdo not always have to be blood samples; buccalepithelial cells, single hairs, epithelial cellsfrom urine, and even fingernails can be used asa source of DNA.2"23 Cigarette butts containsaliva, but only small amounts of DNA (10 to100ng) can be extracted which is insufficientfor Southern blot analyses.24 Saliva on stampsand on the seals of envelopes, although in smallamount (R Decorte, unpublished results), canbe a source of biological material useful in theidentification of persons involved in blackmailor ransom letters. Bones and teeth aresometimes available as evidence of murderscommitted several years in the past or fromhomicides in which the victim was not foundpromptly.2527 After death, human organictissue rapidly decomposes and at the same timeDNA in dead cells degrades under theinfluence of endonucleases and exonucleases.After a few years, only skeletal remains can befound. Therefore, skeletons are important bio-logical samples not only in forensic science butalso in anthropology and archaeology.2829

Set up |PCR reaction

* Laminar flow with UV lamp* Pipettes for non-DNA* Pipettes for DNA

PCRMachine

Figure 3 Laboratory situation and precautions for avoiding contamination in PCRbased typing of genetic polymorphismzs.

Special concerns in PCRThe remarkable sensitivity of the polymerasechain reaction raises special concerns about theaccidental contamination of the DNA samplesor PCR reactions. The most important form ofcontamination is carry over of amplified DNAfrom previous amplifications. As a conse-quence false positive results can be obtainedwhich could lead to incorrect conclusions andin an extreme case might exclude or includesuspects from being involved in a crime. It istherefore of extreme importance to take thenecessary precautions in order to avoid con-tamination (fig 3). This is not only true forforensic laboratories but also for clinical set-tings where PCR is used as a routine dia-gnostic tool. Carry over of PCR products canbe prevented by physical separation of theareas for DNA extraction, setting up of theamplification reactions, and analysis of thePCR products. One should exclude tubes withPCR products from the environment whereDNA extractions are performed. Further-more, separate pipetting devices and the use ofpositive displacement pipettes should help tolimit the possibility of accidental contamina-tion of theDNA samples or the PCR reactions.Additional recommendations have also beenformulated by Kwok and Higuchi.30Another potential source of contamination is

DNA from cells which have been introducedinto the samples either before the biologicalevidence arrived in the laboratory or duringthe extraction procedure. Some experts haveminimised this problem of contamination dur-ing manipulation by stating that the sensitivitylevel (amount ofDNA) at which they performPCR is not enough to detect these contamina-tions.3' However, one should be aware of thepossibility of contamination and take theappropriate measures. In the laboratory,contamination can be avoided in addition tothe physical separation of the DNA extractionby scheduling the extraction of biologicalsamples with low amounts of DNA at a dif-ferent time from the reference samples (bloodsamples) from suspect or victim. Furthermore,the laboratory personnel should be typed andall the results should be checked against theirgenotypes.

Outside the forensic laboratory, it is difficultto exclude contamination of the samples.Therefore, law enforcement agencies have tobe told about the risk of contamination of theevidence with saliva or hair and they have totake the necessary precautions in order toavoid accidental contamination. In a forensiclaboratory, one can control for external con-tamination of the evidence sample by extract-ing some unstained pieces of material of thesame evidence. This unstained material wouldnot contain any biological fluid and thereforeshould be a good negative control. If it gives apositive result one can compare the result firstwith the genetic typings of the laboratorypersonnel in order to exclude internal contam-ination, and then with the typings of thestained material. If no match can be found,contamination by other persons, such as po-licemen, cannot be excluded.

DNAExtraction

* Pipettes for DNA

Analysis ofPCR Products

* Pipettes for PCR products* Gel electrophoresis* Southern blot analysis* Dot-blot analysis

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PCR based DNA typing systemsVNTRs AND MICROSATELLITESIn the past few years, a PCR based method hasbeen developed for a large number of differentpolymorphic loci some of which have beenapplied in forensic investigations (table 3).The majority of these loci are insertion/dele-tion polymorphisms such as VNTRs or micro-satellites. It is not surprising that these loci arethe most popular ones because VNTRs werealready used in conventional DNA typing.Typing of insertion/deletion polymorphismsby PCR involves only amplification and sizefractionation on gel and staining with ethidiumbromide or silver in order to detect the dif-ferent alleles36374647 (fig 2A). However, fordegraded DNA or small samples of DNA it isnot always possible to detect the amplifiedfragments directly after size fractionation.Furthermore, DNA samples from biologicalstains from scenes of crime are often contami-nated with non-human DNA (bacterial or fun-gal) which makes it necessary to quantify theproportion of human DNA.48 One could raisethe number of cycles in order to detect directlythe different amplified alleles with ethidiumbromide. This alternative is, however, notalways applicable for VNTR regions becausean excessive number of amplification cycleswill result in the generation of additional aber-rant fragments, which will interfere with theactual typing. These background fragmentsare thought to be the result of incomplete PCRproducts which act as primers in the additionalcycles.3237 Because of the repeat structure ofthe VNTR region, they can prime at severalsites and give rise to smaller or larger frag-ments differing in a number of repeats fromthe original alleles present in the DNA sample.The number of cycles must therefore belimited in order to avoid such problems.A Southern transfer followed by a hybridis-

ation with a labelled DNA probe can be per-formed in order to detect the amplified VNTRalleles.32 An alternative is the incorporationduring the amplification reaction of a haptensuch as biotin33 or digoxigenin.49 This allowsfor direct detection after Southern transferthrough an intermediate enzyme complex (forexample, avidin-alkaline phosphatase) andchemiluminescence (fig 2B). This method issensitive and rapid and avoids the use of radio-activity. Ultimately, one can incorporate a

Table 3 PCR detection of DNA polymorphisms which have been used in forensicmedicine.

Name HGM 1 Chromosome Heterozygosity Referencelocus localisation %)

p33.4 DlS118 1 67 32 33tp33.6 DISIII 1q23 78 32tpMCT118 DIS80 lp36-p35 81 34 35Apolipoprotein B APOB 2p23-p24 77 36 37 38Gc GC 4q12-q13 * 39DQA1 HLA-DQA1 6p2l.3 82 40 41 42DRB1 HLA-DRB1 6p2l.3 90 41C4A and B C4A; C4B 6p21.3 * 43ABO ABO 9q34. 1-q34.2 * 44c-Ha-ras HRAS I lpl5.5 68 37Type II collagen COL2A1 12q14.3 81 45p3.4BHI IGHJ 14q32.33 74 37pYNZ22 D17S5 l7p13 85 46tp602 DXYS17 Xp22.3/Ypl 1.3 84 1

*Heterozygosity unknown for PCR detectable alleles.tR Decorte et al, in preparation.

fluorescent dye into the PCR products duringamplification followed by the detection of theamplified products with a laser detection sys-tem on an automated sequencer5052 (fig 2C).This system is very sensitive and allows forcomplete automation of the analysis of theDNA samples. Moreover, software is availablefor the determination of the fragment sizes ofthe amplified fragments and output can berouted to database packages for evaluation orstorage. Finally, it is also possible to reamplifythe PCR products with a nested38 or hemi-nested primer pair53 (R Decorte et al, in pre-paration). This way a sufficient quantity ofamplified fragments is obtained for detectionon gel with ethidium bromide. It has also theadvantage of increasing the sensitivity of theassay to a single cell or sperm.53Another problem in PCR typing of VNTR

loci is the efficiency of the amplification ofVNTR regions. Shorter alleles are preferen-tially amplified compared to longer alleleswhich will result in a molar imbalance betweenshort and long allele products.3237 Alleles dif-fering in size by a few hundred base pairs willbe amplified without any problem but whenthe difference is several kb, the longest allelestend not to be detected. Therefore, it is notsurprising that most PCR based typingmethods for VNTR regions have beenreported for a limited number of loci with anallele size of less than 3000 base pairs.

Amplification of microsatellite regions doesnot cause many problems mainly because ofthe smaller size of the region to be amplifiedand the size distribution of the different alleles.Additional amplification products areobserved only for dinucleotide and sometrinucleotide repeats. These fragments areprobably the result of DNA slippage of theTaq DNA polymerase enzyme. Tetra- or pen-tanucleotide repeats do not show these addi-tional amplification products and are thereforemore suitable for forensic investigations andfor presentation in a court room.54

Microsatellites are abundant in the humangenome: dinucleotide repeats occur approxim-ately every 6 kb55 and tri- or tetranucleotiderepeats every 300 to 500 kb.54 The polymor-phism information content of these markers islower than for VNTR regions but because ofthe abundance and the simplicity of amplifica-tion and typing, the same informativeness canbe obtained with a slightly higher number ofloci. Moreover, multiplex PCR or pooling ofdifferent amplification reactions allows forhigh throughput of information as long as theallele size distributions do not overlap. Inaddition, the use of a laser detection systemallows for automation of the analyses.54 Thissystem also alleviates the problem of radio-activity which is currently used for detectionof microsatellites on a sequencing gel.

Because of the small PCR products (100 to500 bp), microsatellites are extremely usefulfor typing degraded DNA samples. While rou-tine forensic DNA samples show some degra-dation, VNTR regions can be typed by PCRalthough they are less efficiently amplified. Forhighly degraded DNA samples, such as DNA

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extracted from bone fragments and teeth,VNTRs cannot be typed because the bulk ofthe degraded DNA is below the allele sizes ofthe VNTR (R Decorte, unpublished results).25Microsatellites and other polymorphic systemswith small PCR products can then be used,usually without any problem.

HLAOther highly polymorphic sequences com-monly used for forensic investigations are thehuman leucocyte antigen (HLA) class II genes(table 3). In the past few years, knowledgeabout the sequence differences which are thebasis for the great diversity among these majorhistocompatibility genes has grown rapidly.This was only possible after the developmentof the polymerase chain reaction technique andrapid techniques for sequencing PCR productswithout going through a cloning step.56 Themolecular basis for the diversity of the majorHLA class II genes is clustered into threehypervariable regions in the second exons ofthe DQA1, DQB1, DRB1, and DPB1 genes.Detection of these differences can be accom-plished by amplification of this exon followedby hybridisation with sequence specific oligo-nucleotides.57 Because of the great variability,it is not always possible to identify each allelewith only one oligonucleotide. Sequence spe-cific oligonucleotides will detect more than oneallele and it is the combination of severaloligonucleotides which will finally identify thealleles and the genotype. Standard dot blot

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Fl C1 Ml1 U M2 C2 F2 Probes- 51

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4 6bl

5 6cl

1 1 52a

12 52b

17 52c

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3 14

6 15

8 16

9 17

10 18

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m m DO m : m m x o DO m m m :VrJ rDj rW w u3 r; m w u w c* * * *C* k * x C

* * *0 0 0 0 C0 0 Oco -4 -4 -4 -j -4 4

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Figure 4 Paternity analysis with the HLA-DRBI system in a family with evidenceof incest. DNA from blood samples was amplified for the second exon of the DRBIgene in the presence of biotin-11-dUTP. Amplified fragments were genotyped with a

reverse dot blot assay. The legal fathers of the two girls could be excluded as thebiological fathers. The uncle of the two girls was shown to be their father. This resultwas confirmed with seven different VNTRs.

typing in which the PCR products are fixed onmembranes and the membranes are hybridisedwith labelled oligonucleotides is a long andlaborious procedure. An alternative approachis the immobilisation of oligonucleotides onthe membranes and hybridisation with thePCR products which were labelled duringamplification.404' This method, which has beentermed the reverse dot blot technique, has theadvantage that only one hybridisation has to beperformed, one for each DNA sample. Fur-thermore, a non-radioactive detection systembased on biotin and alkaline phosphatase fol-lowed by a colorimetric or chemiluminescentsystem is used for the identification of thepositive hybridisation spots.At the end of the 1970s, HLA typing was

introduced in the forensic laboratory for pater-nity testing. This involved mainly the serolo-gical typing of class I genes (A, B, and C) whileHLA-DRB1 has also been used extensively.The DRB 1 gene is the most polymorphicamong the HLA class II genes with 60 knowndifferent alleles.58 An amplification and reversedot blot assay has been developed in our labor-atory and is able to discriminate 52 of the 60possible alleles. With this system, the probab-ility for paternity exclusion is 080 while thepower for discrimination is 0-98. It is thereforeone of the most informative marker systemsbased on PCR detection and analysis. How-ever, for mixed samples, such as the vaginalswabs obtained after rape, it is not alwayspossible to identify each genotype because ofthe complexity of determining the alleles. As aconsequence, the HLA-DRB1 system is notalways as applicable to forensic samples as it isfor paternity determinations (fig 4). TheDQA1 system is less complex and thereforemore interesting for forensic use. The DQA1system was also the first commercial forensicPCR-DNA typing system on the market and isat present probably the most validated PCRsystem in forensic laboratories.425960 However,only nine of the 13 DQA1 alleles are identifiedby this kit with three alleles assigned as a singleallele. Two of these three alleles have a fre-quency of 0 to 5% in white populations whilein North American blacks the two alleles arefound with a frequency of 15% (11th Inter-national Histocompatibility Workshop andConference, Central Data Analysis Commit-tee, Yokohama, 1991). Moreover, a study ofthe HLA-DQA1 diversity in a Hispanic and asouth-east Asian population has shown a slightexcess of homozygotes which could be an indi-cation for the presence of a null allele (an alleleundetectable with the PCR system) or anundiscriminated allele (two different allelesidentified as only one).42 The power of thecommercial PCR kit is probably maximal in awhite population but could be reduced in otherpopulations. The DQA1 system, which wehave developed, is also able to identify nine outof 13 alleles but can distinguish those allelesundiscriminated by the commercial HLA-DQA1 kit. As a consequence, more accuratetyping results can be obtained.4'

55 C

Hybridisationtemperature

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OTHER PCR BASED TYPING SYSTEMSProgress in the identification of genes and theirgene products has also led to the isolation ofthe genes encoding for a number of serologicalproteins and blood groups such as the comple-ment component C4, the group specificcomponent (Gc), and the ABO blood groups.

Elucidation of the genetic differences respon-

sible for the polymorphisms have made itpossible to develop amplification proceduresfor typing different gene products.394344 More-over, genetic typing of genes for which serolo-gical methods have been in use in the forensiclaboratory for decades circumvents the prob-lem of dependability on the expression of thegene. Typing of these polymorphisms can bedone directly by allele specific amplification,by restriction enzyme analyses after PCR, or

by hybridisation with allele specific oligonuc-leotides. Allele specific amplification is a tech-nique which has been developed in recentyears. The goal of this method is the rapididentification of single base polymorphismswithout the need for additional analysis (res-triction digestion or hybridisation) afteramplification. Specific alleles are detected byamplification using a primer with a 3' endwhich is complementary to one of the twoalleles: if the allele is present in the DNAsample an amplification product is obtained,while in the absence of the allele no amplifica-tion is seen. However, this PCR method issometimes difficult to control and it may bemore subject to variation especially in forensiccase samples. Therefore, the transfer fromdevelopment to actual case work should onlybe done after an extensive evaluation periodwith forensic evidence samples. Moreover, theNational Research Council Committee on

DNA Technology in Forensic Science in theUnited States (NRC Committee) has recom-

mended that the allele specific amplificationtechnique should not be used for the analysisof forensic evidence samples.6'

Ultimate proof for inclusion or exclusionmay only be feasible by comparing the nucleo-tide sequences of the two DNA samples.Direct enzymatic sequencing of the PCR pro-

ducts is possible and should be focused on lociwhich have a high degree ofpolymorphism in a

region small enough to amplify and sequencein one step. Because of the diploid nature ofnuclear DNA, a complex pattern of heterozy-gosity at multiple sites in the PCR product andin the sequencing result would be obtained.Mitochondrial DNA, however, contains a re-

gion (control region or D loop) of 1122 basepairs that is composed of two highly variablesegments. The mtDNA has the advantage thatit is a haploid circular DNA segment withmultiple copies present in each cell. Sequencevariation in the variable segments can be deter-mined by PCR and direct sequencing of thePCR products. Rapid and automated sequenc-ing is obtained with a solid phase biotin ap-proach in combination with a fluorescentlabelled sequence primer." The diversityvalue in different populations exceeds 0-95which makes it extremely useful for the deter-mination of maternal lineage and for forensic

purposes.63 In fact, hair samples are very oftenfound at the scene of the crime or on the bodyof the victim or the suspect (table 2). Most ofthese hairs are shed hairs and do not contain ahair root. Without a hair root, it is difficult totype nuclear DNA polymorphisms because thecell nucleus is eliminated during the process ofkeratinisation. In contrast, the hair shafts stillcontain multiple copies of mtDNA.20 More-over, hair samples could have different origins(persons) which makes it necessary to typeindividual hairs.

Procedure for PCR based typingThe analysis of different loci by PCR shouldfit a number of different criteria: (1) the locushas to be very polymorphic, (2) typing by PCRshould be uncomplicated with detection of allpossible alleles, and (3) population genetic data(allele distribution and mutation frequency)should be known. The different loci describedin table 3 are less polymorphic than some ofthe VNTR loci which have been used in con-ventional DNA typing. As a consequence,more loci have to be typed in order to obtainthe same exclusion or inclusion probability.On the other hand, there is a direct relationbetween the mutation frequency and the heter-ozygosity for VNTR loci.64 Although muta-tions have not been described for the differentVNTR loci which can be detected by PCRexcept for D17S5, one should be aware of it inpaternity studies. It is therefore advisable totake a safe approach by not limiting exclusionto only one locus. In forensic evidencesamples, a germline mutation will not interferewith the results because the samples have beenderived from a single person. Somatic muta-tions do occur and could give an exclusion ifone only typed a single cell.The distribution of alleles in the population

may already be known for particular locibecause they have been established previouslyby other methods. However, PCR based typ-ing allows for complete resolution of the dif-ferent alleles on high percentage agarose gelsor polyacrylamide gels.65 Therefore, more ac-curate allele frequencies can be obtained butthis also makes it necessary to redefine thefrequencies in a population with the PCRprotocols.'6 An optimal procedure for theanalysis of forensic evidence or paternity DNAsamples should be based on the polymorphicinformation content of the system and on thecycling parameters for amplification.65 Thiswill ensure that most information is obtainedin a reasonably short time. One could evenamplify two or three different loci simultan-eously but this is only feasible when the allelesize distributions of the loci do not show anyoverlap. In addition, multiplex amplificationshould only be used on DNA samples of goodquality.

Initial analysis with two or three differentpolymorphic loci should give an idea aboutinclusion or exclusion in paternity cases or amatch in forensic cases. An approach with onlythree VNTR loci (DXYS17, DIS111, andD17S5), which have the same cycling para-

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meters, has shown in actual paternity casework that at least one exclusion was observedin 94 7% of the non-paternity samples (RDecorte, unpublished results). A secondexclusion or even a third was observed in76 6% of the non-paternity samples. Thismeans that when the disputed father is not thebiological father of the child, we are able toexclude him in about 75% of the cases withonly three VNTR loci which can be done intwo days.

DNA typing in courtThe application ofDNA technology to foren-sic samples has rapidly become accepted bycourts all over the world. Its potential toexclude wrongly accused suspects in murderor rape cases is not in dispute and is a successcompared to the low exclusion probability ofconventional serology. However, the highinclusion probability of DNA typing hasraised some concern by some biologists andlegal experts.66 Criticism about the use ofDNA technology for the identification of sus-pects has initially focused on the methodologybut it has steadily moved to the level of popu-lation genetics and the statistical evaluation ofthe results."2 Over the past few years, RFLPmethods have been optimised and improved.International cooperation either by forensiclaboratories (for example, EDNAP = Euro-pean DNA Profiling group) or by commerciallaboratories (for example, Cellmark) has led toquality assurance programmes. These pro-grammes are based on standardisation oflaboratory protocols and frequent (every threeto six months) typing of unknown mock casesamples. The ultimate goal is to standardiseDNA profiling (protocols and VNTR probes)which would allow for exchange of data orforensic samples between laboratories. Thebenefit of PCR in this context is that it is easyto transfer the protocols to other laboratoriesand that the way of identifying the differentamplified VNTR alleles can be standardisedby using size markers composed of amplifiedVNTR alleles.'7When two band patterns do not match (for

example, suspect and stain), the conclusionwill be that the suspect/victim could not be thedonor of the stain. In case of a match, the courtwants to know the significance of this event, orwhat the probability is that somebody else hascommitted the crime. In answering thisquestion, the forensic expert calculates thepopulation frequencies of the genotypes forthe different loci. For this reason, the fre-quency of occurrence of the alleles has to beknown. They are usually obtained by typing arandom sample of the population which issufficiently large to provide a good estimate ofthe actual allele frequency. However, theinability to discriminate two large alleles dif-fering in size by one repeat unit is a technicalproblem in conventional DNA typing and ishard to overcome. As a consequence, it is notpossible to obtain a discrete allele frequencydistribution for most VNTR regions if South-ern blot analysis is performed. Instead, a

quasi-continuous distribution is obtained andin order to have allele frequency estimates, it isnecessary to set some arbitrary boundaries forthe band size which has been called 'binning'.67With PCR, however, a discrete allele distribu-tion can be obtained which gives more correctallele frequency estimates.'6 The frequencyestimates are then used to calculate the prob-ability of a match with the assumption that thedata set is in Hardy-Weinberg equilibrium.Deviations from this rule are sometimes ref-lected in the observation of an excess of homo-zygotes compared to the expected number.There could be several explanations for thisphenomenon: (1) the presence of null alleles,(2) incomplete resolution of two closely spacedbut different alleles which has been calledpseudo-homozygosity, or (3) population sub-structure. In forensics, it is appropriate to usereference data on the population from whichthe suspect originated. One genotype could bevery frequent in a white population but rare ina black population. This will influence thesignificance of a match by a factor 10 to 100. Ifthe reference data have been obtained from amixed population then the difference betweenthe exact population and the mixed populationfrequencies would be even less pronounced.'368Nevertheless, it is advisable to take a conserva-tive approach and calculate the probability of amatch by using p (= allele frequency estimate)or even a 2p statistic for a single band patterninstead of p2. One should also not take intoaccount the alleles shared with the other per-sons (suspects or victim) who are involved inthe case. All these methods will ensure that thefrequency estimates are biased in favour of thesuspect.

Finally, to overcome population substruc-ture or ethnic background of a subject, theNRC Committee has recommended the use ofthe 'ceiling principle': the highest frequencyfound in 15 to 20 different populations (each100 persons typed) would be used or 5%.69Recently, the NRC proposal has come underattack by population geneticists and statisti-cians arguing that there is no scientific basis forthe 'ceiling principle' and that it is built onerroneous assumptions about population gen-etics (subpopulations within ethnic groupsshow a higher diversity than the majorgroup).70 It also does not take into account thefact that the suspect may not belong to theethnic group of which the allele frequencyestimate has been used to calculate the signific-ance of the match. An alternative approachwould be to use a likelihood ratio (LR) whichis simply the inverse of the genotype probabil-ity.7' The LR is also comparable with thepaternity index (PI) which is frequently usedfor evaluating disputed paternity cases.

Future perspectivesTECHNOLOGYAutomation of the PCR process has been oneof the first innovations after the introductionof PCR technology. Additional automation isat this moment under development firstly insize separation of the amplified fragments,

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secondly in the preparation of PCR reactions,and thirdly in sequencing. Robotic workstations can prepare the PCR reactions in a 96well microplate format including the additionof the DNA samples. Sequencing of the PCRproducts can be taken over by automaticsequencing robots and finally automatic load-ing devices can load the sequencing reactionsor the amplified PCR products onto automaticsequencers. The role of the laboratory person-nel is limited and involves only programmingof the robot, supplying the correct solutions,and evaluation of the results.

ADDITIONAL MARKERSSome of the most promising markers are theminisatellite variant repeats (MVR). Jeffreys etall2 have observed that not only the number ofrepeats of minisatellite or VNTR regions canbe different but also that the repeats them-selves can be polymorphic at the nucleotidelevel. These polymorphisms are sometimesdetectable by restriction enzymes but also byallele specific amplification. It is possible thattwo alleles of the same length can have adifferent composition in repeat nucleotide se-quence. This has important consequences forforensic analysis when one uses only a singleVNTR locus or when one only has a resultwith one polymorphic marker: the matchingband pattern could be an exclusion if one knewthe exact base composition of the matchingalleles. Analysis of the internal variation eitherby restriction digestion or by allele specificamplification is independent of artefacts likeband shifting which occurs sometimes in con-ventional DNA typing. Side by side compari-son is not necessary with this method as is thecase with current technology for the analysis ofVNTRs. Moreover, the patterns generated canbe stored as a binary (1 =present, 0 =.absent)code which suggested the name 'digital DNAtyping' for this method.73 It allows for easycomparison in large databases because it doesnot have to take into account size measurementerrors. The efficiency for paternity testing is,however, limited by the high mutation rate atthe minisatellite loci which have these internalvariations. Furthermore, as explained before,allele specific amplification is more subject tovariation in experimental conditions andshould therefore only be used after extensiveevaluation.

ConclusionsThe polymerase chain reaction technique hasbecome a valid alternative to Southern blottingfor the detection of highly polymorphic locisuch as variable number of tandem repeats. Ithas not only facilitated the analysis of geneticpolymorphisms but it has also opened the wayfor the use of new markers such as microsatel-lites and the mitochondrial control region.Because of the simplicity of the protocols, itallows for standardisation and exchange ofdata with other forensic laboratories. Thehigher sensitivity and the increased resolutionof small size differences increases the number

of cases in which a result can be obtained andallows for more accurate statistical calculation.PCR based typing of forensic case samplesshould, however, be performed with the stron-gest precautions against contamination. Thiswill ensure that forensic DNA analysis is relia-bly performed to the highest standard.

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