DNA profiling 2 - Science at St. Mary'sscienceatstmarys.weebly.com/.../2.5.4_dna_profilling.pdfDNA...

2DNA profiling UN

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European Initiative for Biotechnology Education

Contributors to this UnitDorte Hammelev (Unit Co-ordinator)

Dean Madden, Søren Nørby, Jill Turner

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2 UNIT 2: DNA PROFILING EIBE European Initiative for Biotechnology Education 1998

EIBE Co-ordinatorHorst Bayrhuber, Institut für die Pädagogik der Naturwissenschaften an der Universität Kiel, Olshausenstraße 62,D-24098 KIEL, Germany. Telephone: + 49 (0) 431 880 3166 (EIBE Secretary: Regina Rojek). Facsimile: + 49 (0) 431 880 3132.

EIBE ContactsAUSTRIA❙ Rainhart Berner, Höhere Bundeslehr- und Versuchsanstalt für Chemische Industrie Wien, Abt. für Biochemie,Biotechnologie und Gentechnik, Rosensteingasse 79, A-1170 WIEN.

BELGIUM❙ Vic Damen / Marleen Van Strydonck, R&D Groep VEO, Afdeling Didactiek en Kritiek, Universiteit Antwerpen,Universiteitsplein 1, B-2610 WILRIJK.

DENMARK❙ Dorte Hammelev, Biotechnology Education Group, Foreningen af Danske Biologer, Sønderengen 20, DK-2860 SØBORG.❙ Lisbet Marcussen, Biotechnology Education Group, Foreningen af Danske Biologer, Lindevej 21, DK-5800 NYBORG.

EIRE❙ Catherine Adley / Cecily Leonard, University of Limerick, LIMERICK.

FRANCE❙ Gérard Coutouly, LEGTP Jean Rostand, 18 Boulevard de la Victoire, F-67084 STRASBOURG Cedex.❙ Laurence Simonneaux / Jean-Baptiste Puel, Ecole Nationale de Formation Agronomique, Toulouse-Auzeville,Boîte Postale 87,F-31326 CASTANET TOLOSAN Cedex.

GERMANY❙ Horst Bayrhuber / Eckhard R. Lucius / Regina Rojek / Ute Harms / Angela Kroß, Institut für die Pädagogik derNaturwissenschaften an der Universität Kiel, Olshausenstraße 62, D-24098 KIEL.❙ Ognian Serafimov, UNESCO-INCS, c/o Jörg-Zürn-Gewerbeschule, Rauensteinstraße 17, D-88662 ÜBERLINGEN.❙ Eberhard Todt, Fachbereich Psychologie, Universität Gießen, Otto-Behaghel-Straße 10, D-35394 GIEßEN.

ITALY❙ Antonio Bargellesi-Severi /Alessandra Corda Mannino/ Stefania Uccelli , Centro di Biotecnologie Avanzate,Largo Rosanna Benzi 10 , I-16132 GENOVA.

LUXEMBOURG❙ John Watson, Ecole Européenne de Luxembourg, Département de Biologie, 23 Boulevard Konrad Adenauer,L-1115 LUXEMBOURG.

THE NETHERLANDS❙ David Bennett, Cambridge Biomedical Consultants, Schuytstraat 12, NL-2517 XE DEN HAAG.❙ Fred Brinkman, Hogeschool Holland, Academy for Communication, Postbus 261, NL-1110 AG DIEMEN.❙ Liesbeth van de Grint / Jan Frings, Hogeschool van Utrecht, Educatie Centrum voor Biotechnologie, FEO,Afdeling Exacte Vakken, Biologie, Postbus 14007, NL-3508 SB UTRECHT.

SPAIN❙ María Sáez Brezmes / Angela Gómez-Niño / Rosa M. Villamañán, Facultad de Educación, Universidad deValladolid, Geologo Hernández Pacheco 1, ES-47014 VALLADOLID.

SWEDEN❙ Margareta Johansson, Föreningen Gensyn, PO Box 37, S-26881 SVALÖV.❙ Elisabeth Strömberg, Östrabo Gymnasiet, S-45181 UDDEVALLA.

THE UNITED KINGDOM❙ Wilbert Garvin, Northern Ireland Centre for School Biosciences, NIESU, School of Education, The Queen’s University ofBelfast, BELFAST, BT7 1NN.❙ John Grainger / John Schollar / Caroline Shearer, National Centre for Biotechnology Education, The University of Reading,PO Box 228, Whiteknights, READING, RG6 6AJ.❙ Jill Turner, Department of Science and Technology Studies, University College London, Gower Street, LONDON, WC1 6BT.❙ Paul Wymer, Society for General Microbiology, Marlborough House, Basingstoke Road, READING RG7 1AE.

The European Initiative for Biotechnology Education (EIBE) seeksto promote skills, enhance understanding and facilitate informedpublic debate through improved biotechnology education inschools and colleges throughout the European Union (EU).

EIBE European Initiative for Biotechnology Education 1998 3UNIT 2: DNA PROFILING

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2UNIT

DNA profiling

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Contents★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

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European Initiative for Biotechnology Education

❙ Development team,Copyright, Safety 4

❙ DNA profilingThe background 6The technology 10

❙ Classical profiling 12Examples 14

❙ Modern profiling 16

❙ Reliability 20

❙ Discussion 24

❙ References 25

❙ Practical activityFingerprint simulation 26

Edition 2/2 completed January 1998

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EIBE contributors● Dorte Hammelev (Unit Co-ordinator)

Frederiksberg HF kursusCopenhagen, Denmark

● Dean MaddenNCBE, The University of ReadingU.K.

● Søren NørbyLab of Biological AnthropologyInstitute of Forensic MedicineCopenhagen University, Denmark

● Jill TurnerDept. of Science and TechnologyStudies, The University of LondonU.K.

Design, illustration and typesetting: DeanMadden and Caroline Shearer, NCBE, TheUniversity of Reading, RG6 6AJ

AcknowledgementsWe are very grateful to Niels Morling,Hanna Hansen and Birthe Eriksen, all ofthe Department of Forensic Genetics,University of Copenhagen, especially forthe provision of photographs and researchdata.

The practical activity in this unit wasdeveloped by John Schollar at the NCBE.

Dorte Hammelev (Frederiksberg HFKursus, København, Denmark), WilbertGarvin (Northern Ireland Centre forSchool Biosciences, The Queen’s Universityof Belfast, The United Kingdom), JohnSchollar (National Centre forBiotechnology Education, The Universityof Reading, The United Kingdom) and JillTurner (University College, London, TheUnited Kingdom) arranged and ran amultinational workshop in which thematerials in this Unit were tested. EIBE

would like to thank them and the teachersfrom Denmark, Eire and Germany whotook part and gave many helpful commentson the draft materials.

The workshop participants were:

From Denmark: Lisbet Leonard; LeneTidemann; Mario Bro Hassenfeldt; GretaGrønqvist; Jytte Jørgensen; Tine Bing; PerVollmond; Anker Steffensen.

From Eire: John Lucey; Michael O’Leary;Bruno Mulcahy; Tim O’Meara; TomMoloney; Brendan Worsefold; Frank Killelea.

From Germany: Ulrike Schnack; WernerBährs; Jürgen Samland; Cristel Ahlf-Christiani; Erhard Lipkow; Hubert Thoma.

From the EIBE team: Eckhard R. Lucius;Catherine Adley; Jan Frings; Wilbert Garvin;Jill Turner; Dean Madden; John Schollar;Dorte Hammelev.

© CopyrightThis EIBE Unit is copyright. Thecontributors to this Unit have asserted theirmoral right to be identified as copyrightholders under Section 77 of the Copyright,Designs and Patents Act, UK (1988).

Educational use. Electronic or paper copiesof this EIBE Unit, or individual pages fromit may be made for classroom use, providedthat the copies are distributed free-of-charge or at the cost of reproduction, andthe contributors to the unit are credited andidentified as the copyright holders.

Other uses. The Unit may be distributed byindividuals to individuals for non-commercial

purposes, but not by means of electronicdistribution lists, mailing (listserv) lists,newsgroups, bulletin board or unauthorisedWorld Wide Web postings, or other bulkdistribution, access or reproductionmechanisms that substitute for asusbscription or authorised individual

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SafetyIn all of the EIBE Units, we have tried tocheck that all recognised hazards have beenidentified and that suitable precautions aresuggested.

Where possible, the proposed proceduresare in accordance with commonly-adoptedgeneral risk assessments. If a special riskassessment may be necessary, this has beenindicated.

However, users should be aware that errorsand omissions can be made, and thatdifferent employers and educationalauthorities adopt different standards.Therefore, before doing any activity, usersshould always carry out their own riskassessment. In particular, any local rulesissued by employers or educationalauthorities MUST be obeyed, whatever issuggested in the EIBE Unit.

Unless the context dictates otherwise, it isassumed that:

● practical work is carried out in aproperly equipped and maintainedscience laboratory;

● any mains-operated equipment isproperly maintained;

● care is taken with normal laboratoryoperations such as heating substances;

● good laboratory practice is observedwhen chemicals or living organisms areused;

● eye protection is worn whenever thereis any recognised risk to the eyes;

● pupils and/or students are taught safetechniques for activities such ashandling chemicals andmicroorganisms.

access, or in any manner that is not anattempt in good faith to comply with theserestrictions.

Commercial use. Should you wish to usethis material in whole or in part forcommercial purposes, or to republish it inany form, you should contact:

EIBE Secretariatc/o Institut für die Pädagogikder NaturwissenschaftenUniversität KielOlshausenstraße 62D-24098 KielGermany

Telephone: + 49 431 880 3166Facsimile: + 49 431 880 3132E-Mail: [email protected]

About this UnitThese materials have been devised bypractising teachers and educationalists fromseveral European countries, broughttogether with financial support andencouragement from DGXII of theEuropean Commission, under the auspicesof EIBE, the European Initiative forBiotechnology Education.

The EIBE materials have been extensivelytested in workshops involving teachersfrom across Europe.

The views expressed in this Unit and theactivities suggested herein are those of theauthors and not of the EuropeanCommission.

Particular attention should be paid to thegeneral safety guidelines given in theintroduction to this Unit, and to the specificsafety guidelines throughout the text.

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IntroductionThe analysis of human DNA hasapplications, apart from in pure research, intwo main areas.1. Health care: including diagnosis of

hereditary disease, chromosomeaberrations and cancer.

2. The judicial system: identification ofsuspects in criminal cases (especiallymurder, rape and other violent assaults)and analysis of family relationships indisputed paternity and immigrationcases.

The teaching material in this unit dealsmainly with the applications of DNAanalysis within the judicial system.

A DNA analysis carried out to clarify a legalproblem, be it a criminal case or a questionof disputed family relations, only deals witha tiny part of the human genome. Inessence, the routine analysis consists of thedetermination of the lengths of four or fiveselected chromosomal DNA segments. Theresult is an individual combination ofletters and numbers called a DNA profile.Some have compared the DNA profile to abar code. In fact, the original method,developed for legal purposes, gave its resultas a series of dark bands on an X-ray film.This was called ‘genetic fingerprinting’. Themethod which is used today, however, iscarried out differently. It is called DNAprofiling.

Application of DNA profilingEvery day newspapers feature stories ofmurder and rape, and DNA profiling is nowroutinely used in solving these cases. DNAprofiles of blood or semen stains from thescene of the crime are compared with thoseof possible suspects.

In cases of disputed paternity, an initialcomparison between the DNA profiles of amother and child will show which parts of

the child’s profile must have beencontributed by the father. DNA analysis ofthe putative father makes it possible todecide if the man in question may be thechild’s father or could not be, i.e. if hisDNA profile is compatible with the part ofthe child’s profile not contributed by themother. This can also be applied toimmigration cases when the familyconnection between an established citizenand a new immigrant requesting citizenshipon the basis of family relationships, may beestablished or disproved.

DNA profiling may also be used to aid theidentification of bodies, by comparisonwith possible relatives. It has provedvaluable in sea and air accidents whenvictims are beyond recognition (1). It hasalso been used for victims of terrorism inIsrael and the former republic ofYugoslavia, and of war, e.g. in the Gulf warin 1990.

Identification of long dead human remainsis also now possible due to the developmentof methods allowing the extraction ofDNA from bones, including teeth. This hasallowed identification of the remains ofpast victims of crime and has been used inmore notorious cases such as the Nazidoctor Mengele and the last Russian tsarand his family (2,3).

In the case of the Russian tsar it waspossible, about 75 years after the executionof the family, to clarify which of the nineskeletons that were found in the mass gravein question came from the body of the tsar,his wife and three of their children.Similarly it was possible by DNA analysis toprove that a woman who throughout heradult life had claimed to be Anastasia, theyoungest of the daughters of the tsar andhis wife, had been an impostor (see page23)(4).

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DNA profiling - the background★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★


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Archaeologists and anthropologists havealso found uses for DNA analysis. Astunningly successful example is the recentanalysis of DNA from the prehistorichuman skeleton found in Neanderthal nearthe German town of Düsseldorf in 1856.This skeleton is estimated to be between30,000 and 100,000 years old and representsan extinct group of human beings. Theresults of the DNA analysis show that theNeanderthals and modern man, who duringseveral thousand years coexisted in Europeand elsewhere, are two separate specieswhose most recent common ancestors livedabout 500,000-700,000 years ago (5).

DNA analyses are also being used in thestudy of viruses, bacteria, plants andanimals. They have proved valuable both inresearch (i.e. to elucidate relationshipsamong species) and for more practicalpurposes such as diagnosis of infectiousdiseases, tracking stolen or illegallytransported animals, and surveillance ofgenetically manipulated plants.

The human genomeMost of the human genome is located incell nuclei (nuclear DNA). A small, butimportant and interesting part, is found inthe mitochondria (mitochondrial DNA).This will be dealt with separately.

Nuclear DNAIn man the nuclear DNA comprises 46 longmolecules, each of which forms ‘the geneticbackbone’ of a chromosome. The 46chromosomes of each cell consist of 23pairs, one chromosome from each pairhaving been inherited from the individual’smother the other from the father. This isthe basis of Mendel’s universal first law ofinheritance and means that it is possible byDNA analysis to trace the biologicalrelations within a family, including thesegregation of disease genes.

One pair of the 23 pairs of chromosomes,the sex chromosomes, differ between thetwo sexes: two X chromosomes (XX) in the

female, an X and a Y (XY) in the male.Analysis of blood stains and tissue samplesfor the presence of Y-chromosomal DNAcan therefore be used to identify the sex ofthe donor. The remaining 22 pairs ofchromosomes are called autosomes.

It is estimated that the human nucleargenome has between 50,000 and 100,000genes which code for the many differentproteins of the organism. However, only aminor part of the nuclear DNA containssuch coding sequences. The non-codingsequences are located within individualgenes, forming intervening sequences orintrons, as well as between genes. The DNAsequences used in DNA profiling belong tothe non-coding sequences. The complexityand variation of genetic material means thateach human being, other than monozygousindividuals (identical twins, triplets etc.), hasa unique genome and thus a personal DNAprofile.

MutationsDNA is stable but not static. Occasionallymutations happen in DNA, changing thesequence of base pairs. A mutation canhave serious consequences if it changes thesynthesis or function of an importantprotein. However, most mutations have noimmediate consequences for the organism,as they take place in non-coding sequences,either within or outside a gene. Suchharmless, neutral, mutations form the basisfor most of the differences betweenindividual genomes.

Highly variable regionsIn routine forensic DNA profiling, regionsof the genome are analysed in which aspecific sequence of base pairs is repeated avariable number of times in a continuoussequence (Fig. 1). These regions are calledVNTR regions, VNTR being anabbreviation of variable number of tandem

repeats.

The length of a given VNTR region of acertain chromosome will thus depend on

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the number of base pairs in the repeatsequence and the number of times it isrepeated. In DNA profiling the lengths of anumber of different VNTR regions aredetermined and the DNA profile is thus asimple combination of these lengthsexpressed in a format agreed upon by theinternational forensic genetics community.

The discovery of VNTR regionsThese especially variable regions of thegenome were discovered by professor AlecJeffreys and his co-workers at the Universityof Leicester, UK. They were studying thegene coding for myoglobin, the red oxygen-binding protein in muscle cells. During thiswork they discovered that one of theintrons in this gene contains a sequence ofbase pairs tandemly repeated a number oftimes and that the number of repeats canvary among myoglobin genes and thusamong individuals. Jeffreys and his groupwanted to use this variable region as amarker in their effort to find thechromosomal location of the myoglobingene. They therefore isolated a piece ofDNA containing this region and used it as aprobe in DNA analyses.

A probe is a DNA molecule that can base-pair, or hybridise, with the DNA strands inthe gene of interest. The probe is labelled,either chemically or radioactively, so that itcan be detected in the analysis in which it isbeing used, e.g. by means of a sensitivephotographic film (see Fig.5 ).

When Jeffreys and his co-workers used theprobe they discovered to their surprise that

every analysed sample resulted in numerous‘bands’ on the photographic film, due tothe fact that the probe had bound to a largenumber of DNA regions from each person.The band pattern turned out to be verypersonal and thus ‘the genetic fingerprint’was discovered. Later the various VNTRregions causing the many bands wereidentified and characterised and it becamepossible to analyse them separately which isthe principle behind today’s DNA profiling.

Alec Jeffreys has received muchrecognition, including a knighthood, forthis discovery and its subsequentdevelopment, laying the grounds forforensic DNA analyses. His work is one ofmany instances of an important discoverybeing made during an investigation withquite a different goal. It also shows howbasic research can make discoveries whichcan quickly have valuable practicalapplications.

Mitochondrial DNAMitochondrial DNA (mtDNA) is a small,circular DNA molecule with about 16,600base pairs. In comparison nuclear DNAconsists of some 6 billion base pairs or

Figure 1. A VNTR region in a chromosomepair: the two chromosomes have 10 and 18repetitions respectively .

paternal chromosome:

maternal chromosome:

Figure 2. Sir Alec Jeffreys


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about two metres of DNA! As eachmitochondrion contains 5-10 mtDNAmolecules, and a cell can have hundreds oreven thousands of mitochondria, the cellsfrom an individual will, on average, containthousands of mtDNA molecules withidentical sequences of base pairs. This highcopy number per cell explains why mtDNAis the part of the genome which is mostlikely to be recovered from ancient remainsand from biological stains.

As mtDNA is located outside the cellnucleus, it is inherited exclusively throughthe female, the sperm only contributingnuclear DNA. This means that a woman,her children, mother, maternalgrandmother, and other family memberslinked through an unbroken female linehave identical mtDNA sequences.Mitochondrial DNA analysis is thereforeuseful in identifying biological relationshipseven across a separation of manygenerations, provided there is an unbrokenfemale line.

Mutations can occur in mtDNA, as innuclear DNA, and per base pair they do soat a higher rate. Mitochondrial DNA is lessprotected against damage and does nothave access to the DNA repair mechanisms

present in the nucleus. Over millennia asignificant variation in the mtDNAsequence of man has therefore arisen.About half of that variation is located intwo minor, non-coding regions of themolecule and sequence analysis of thesehighly variable regions have become animportant tool in both forensic andanthropological investigations. It wasmtDNA analyses that made it possible toidentify the skeletons of the last Russiantsar and his family (2,3) and reveal theidentity of ‘the false Anastasia’ (4). It wasalso mtDNA analyses that gave us the firstimportant genetic information about theNeanderthals revealing that they were ahuman species only remotely related tomodern man (5).

More about VNTR regionsMany of the VNTR regions that were first discovered had a rather large number of base pairs (typically20-50) per repeat and a variation in the number of repeats from 50 to several hundred. Such a regioncan thus vary from 1,000 to 10,000 base pairs or more. The number of repeats, and thus the length ofthe VNTR region, is a characteristic which is inherited according to the fundamental Mendelian principleof segregation. In genetic terms a VNTR region is therefore a locus with a number of alleles, eachcharacterised by a certain length. Such a locus, with a variety of alleles each showing a fairly high frequency,is said to constitute a genetic polymorphism (polymorphism = the occurrence of many forms; here:variants). In a highly variable VNTR locus more than 95 % of the population will have alleles of differentlengths and thus be heterozygous in this locus, and the likelihood that two unrelated individuals havethe same combination of alleles in a given VNTR locus is often much smaller than 1 %.

More recently, a vast number of VNTR regions have been identified which have repeated sequences ofonly 2-4 base pairs, showing a variation of, for instance, 5 to 15 repeats. A number of these so-calledSTR regions (short tandem repeats) form the basis of modern advanced forensic DNA profiling.

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DNA profiling - thetechnology★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Two types of DNA profiling, classical

and modern, will be described. Both use

electrophoresis as an essential element

of the technique so it is first necessary

to have a clear understanding of the

electrophoresis process.

ElectrophoresisElectrophoresis means migration in anelectric field. As DNA molecules containnumerous phosphate residues they arenegatively charged when in an alkalinesolution. In an electrical field they thereforemigrate towards the positive electrode, theanode. If this migration takes place in a gel,the DNA molecules are at the same timeseparated according to size, as the smallmolecules move faster through the mesh ofthe gel than larger ones. The gel effectivelyacts as a molecular sieve. Gel electro-phoresis is a simple and extremely usefultechnique for separation of DNAmolecules according to size, and it is used inmany forms of DNA analysis.

Classical DNA profilingBefore reading any further, look at pages 12-13 for an outline of the procedures involvedin classical DNA profiling.

Classical DNA profiling deals with largerVNTR regions and is carried out as anRFLP (restriction fragment length polymorphism)analysis. This term originated in the 1970’swhen it was discovered that human DNA,when treated with restriction enzymes, iscut into smaller molecules called restrictionfragments.

After treatment with a restriction enzyme aparticular autosomal VNTR region from asingle individual will usually result inrestriction fragments of two different sizes,one from the maternal and the other from thepaternal chromosome (Fig. 3).

It was mentioned earlier that the alleles of aVNTR locus are elements in a geneticpolymorphism. As the polymorphic allelesused in classical DNA profiling are definedby the lengths of the correspondingrestriction fragments, this type ofpolymorphism is called restriction fragmentlength polymorphism (RFLP).

Restriction enzymes - fundamentaltools in DNA analysisRestriction enzymes are found in bacteria.They are characterised by their ability torecognise a specific sequence of usually 4-6base pairs in a DNA molecule and ‘cut’ thetwo DNA strands either within or close to therecognition sequence. In precise termsrestriction enzymes are sequence-specificDNA endonucleases. Consequently, by usinga restriction enzyme, it is possible,reproducibly, to ‘cut’ long DNA moleculesinto well defined fragments. They havetherefore become biochemical tools offundamental importance in both genetechnology and DNA analysis.

Figure 3. Two VNTR alleles (see Fig.1) showingthe sites of action of a restriction enzyme: thelength of the resulting DNA fragments of interest isindicated by the distance between the cuts (arrows).

paternal chromosome:

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maternal chromosome:

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e e e5’-GAATTC-3’ 5’-GANTC-3’ 5’-GGCC-3’3’-CTTAAG-5’ 3’-CTNAG-5’ 3’-CCGG-5’

EcoRI HinfI HaeIII

Figure 4. Recognition sequences and ‘cuttingpattern’ of three restriction enzymes: mostenzymes have symmetric (palindromic) recognitionsequences.

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Multiple VNTR site locationIf the quality of the band pattern on theautoradiogram, i.e. the exposed film, isgood, the membrane is used forhybridisation with another proberecognising another VNTR region. Thefirst probe is removed, usually by boilingthe membrane. This procedure does notchange the pattern of the bound single-stranded restriction fragments nor theircapacity to base-pair with a new probe.Steps 5-7 can then be repeated using aprobe which binds to another VNTRregion. The membrane is usually reused inthis way for analysis of the four or fiveVNTR loci included in DNA profile. It is,of course, a great advantage, both forsimplicity and accuracy, that all loci can beanalysed in a single gel electrophoresis run.

Advantages and limitationsThe greatest advantage of RFLP basedDNA profiling is the high degree ofvariability in the classical VNTR regions. Inother words there are a large number ofalleles for each locus. Consequently it isextremely unlikely that the DNA profiles oftwo unrelated individuals will be identical.There is, however, an inherent weakness inthis analysis due to the fact that therestriction fragments are rather large andtherefore show a rather continuous size

distribution after electrophoresis (i.e. thebands may not be clearly separated). Thismakes it difficult to distinguish betweenfragments of similar, but different lengths,especially if the samples in question havebeen analysed on separate gels. Two gelsgiven the same treatment may show slightlydifferent results after electrophoresis ontwo different sets of equipment, or evenwhen using the same equipment at differenttimes.

At least 20 nanogram of purified and fairlyintact DNA is needed for RFLP-basedDNA profiling. This limits the usefulnessof this method in criminal case work whenthere may be very little, rather degradedDNA from extracts of specimens such asblood or semen stains. Further problemsmay be caused by other compounds in theextracts, e.g. dyes from cloth, such as theblue colour of denim, which may stick tothe DNA and influence the migration rateof the fragments, giving misleading results.This might cause the DNA profile of astain to differ from that of a blood sampledrawn from the same person, and theperson to be wrongly excluded from theinvestigation. However, a consistent minordifference in migration of fragments from allanalysed VNTR regions will alert theexperienced investigator to this problem.

More about restriction enzymesBacteria defend themselves against attacks from bacterial viruses (bacteriophages or simply: phages) withrestriction enzymes. The enzymes cut invading viral DNA and thus render it harmless. In this way theyrestrict the spectrum of viruses which can infect a particular bacterium, hence their name: restrictionenzymes. (The bacterium protects its own DNA against the restriction enzyme by means of another enzymewhich puts a methyl group on one of the bases in the recognition sequence, wherever it might occur in thebacterial genome. This modification prevents the sequence from being recognised by the restriction enzyme.)

Several hundred restriction enzymes, each with different recognition sequences are now known and can bebought from molecular biological suppliers. The enzymes are given italicised three-letter ‘names’ which areabbreviations of the name of the bacterial species in which the enzyme was originally found, e.g. EcoRI(pronounced ‘ecco are one’) was originally found in the intestinal bacterium Escherichia coli, better known asjust E. coli. Other examples (Figure 4) are HinfI (pronounced ‘hin ef one’) from Haemophilus influenzae andHaeIII (pronounced ‘hay three’) from Haemophilus aegypti. In Europe HinfI is traditionally used in classicalDNA profiling whereas HaeIII is used in the U.S..

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Samples are received. DNA is extractedand purified. In blood only the white bloodcells contain both nuclear andmitochondrial DNA. (In mammals,including man, the red blood cells do notcontain DNA; platelets contain onlymitochondrial DNA).

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The DNA samples are treated with therestriction enzyme HinfI which cuts theDNA in several places including each sideof the VNTR regions of interest.

The samples containing the restrictionfragments produced in step 2 are pipettedinto individual wells in an agarose gelimmersed in an alkaline buffer (in anelectrophoresis tank). Electrophoresis isstarted.

After electrophoresis the gel is removed.DNA fragments are transferred from the gelto a nylon membrane by placing themembrane on top of the gel and covering itwith a layer of absorbent paper towels. Thisis called Southern blotting (after thebiochemist, Ed Southern). The gel is treatedwith a strong alkaline solution (NaOH) todenature the DNA fragments (break thehydrogen bonds between the DNA strands)so that the fragments are bound to themembrane as single strands.

The nylon membrane with the bound butinvisible pattern of single-stranded DNAfragments is submerged in a solutioncontaining a radioactive, single-strandedDNA probe. The nucleotide sequence ofthe probe is complementary to part of thesequence of the VNTR region to becharacterised. The strands of the probehybridise with the membrane bound,single-stranded fragments containing thecorresponding VNTR region.

The nylon membrane is lifted out of thehybridisation solution, unbound probe iswashed off and the membrane is dried. It isthen placed in contact with a sensitivephotographic film which will reveal whereon the membrane the radioactive probe isbound and hence the positions of thefragments of interest.

The film is developed and ‘bands’corresponding to the restriction fragmentsof interest appear. The approximate sizesof the fragments can then be determinedfrom their distance of migration duringelectrophoresis, by comparison with DNAmolecules of known size run in a separatelane on the gel.

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Figure 5. Classical DNA profiling - the steps of an RFLP analysis

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Problem no. 1: Disputed paternityFigure 6 shows the result of an RFLP-basedDNA profile in a case of disputed paternity.In interpretation of the result keep basicgenetic principles in mind: a VNTR locushas been analysed in which every individualhas two alleles, a paternal and a maternalone.

1. In this case two possible fathers havebeen presented. Which of the followingquestions can be answered with thehighest degree of certainty? (Givereasons for your answer).a) Which man is the child’s father?b) Which man cannot be the child’sfather ?

2. Why are up to five VNTR regionsroutinely analysed in cases of disputedpaternity ?

Problem no. 2: ImmigrationFigure 7 shows the DNA profile of a familywhere two VNTR regions have beenanalysed. The story is that a family ofrefugees with three children have been givenasylum. Later, an adolescent boy arrives andasks for permission to be granted residencetoo. Both he and the family claim that he isthe fourth child of the family. Theauthorities ask for DNA analysis in order toclarify the biological relationship.

1. Can the adult couple be the parents ofchildren numbers 1, 2 and 3 ? (Givereasons for your answer.)

2. Can the adult couple be the parents ofchild number 4 ? (Give reasons for youranswer.)

3. Give your opinion, based on youranswers to the above questions, ofwhether child no. 4 should be grantedresidence in the country on the basis ofhis relationship to the family. (See alsothe questions for discussion on p. 24).

Case-oriented examples★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

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Lane:1 - Mother2 - Child3 - Father A?4 - Father B?

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Figure 7.

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Problem no. 3:Figures 8 and 9 show the distribution of alleles of the two VNTR loci D1S7 and D5S43 in aEuropean population.

1. Describe in words what each graph shows.

2. Which allele is the most frequent in each of the two loci ?

3. Are these two VNTR regions equally useful for DNA analysis ina) a murder case,b) a case of disputed paternity andc) an immigration case ? (Give reasons for your answer.)

Figure 8. VNTR locus D1S7 (probe: MS1).

frequency%

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The abscissa shows allele size; the ordinate shows the frequency of the alleles as a % of thoseanalysed. The repeat sequence consists of nine base pairs. The smallest alleles have a little morethan 130 repeats, the largest ones over 2,000. The mutation rate of this locus is ca. 5 %, which isrelatively high, even for VNTR regions. D1S7 is located on chromosome 1q, i.e. the long arm ofchromosome 1. (Source: Cellmark sales catalogue)

Figure 9. VNTR locus D5S43 (probe: MS8).

The abscissa shows allele size; the ordinate shows the frequency of the alleles as a % of thoseanalysed. This repeat sequence consists of 30 base pairs. The smallest alleles have about 80repeats, the largest ones have several hundred. (Source: Cellmark sales catalogue)

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2 4 6 8 10 12 14 16 18 20 kballele size kb (1 kb = 1000 base pairs)

2 4 6 8 10 12 14 16 18 20 kballele size kb (1 kb = 1000base pairs)

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Modern forensic DNA profiling deals withthe analysis of STR regions (short tandemrepeats, see information box on page 9) wherethe repeated sequences are four base pairs long(see Fig. 11). Before any determination of thelengths of the alleles, the regions in questionare amplified using the PCR (polymerasechain reaction) technique, a very efficientmethod for in vitro replication of DNA.Under suitable conditions, this techniquemakes it possible to produce several millioncopies of a specific DNA sequence within afew hours.

Polymerase chain reaction (PCR)In order to carry out DNA amplification bythis method the following are necessary:DNA from the sample to be analysed, DNApolymerase (an enzyme which synthesizescopies of pre-existing DNA), and the fourdeoxyribonucleoside triphosphates, i.e. the‘building blocks’ for the new DNA molecules.Two small, single-stranded DNA molecules,20-30 nucleotides long, are also needed. Theseoligonucleotides are designed to hybridise, or

base-pair, to the DNA on each side of theregion to be amplified (see Fig. 10). Theseoligonucleotides, called primers, form thestarting points for synthesis of new DNAstrands, i.e. they initiate polymerisation. PCRamplification of a DNA sample consists ofthe following steps which are repeated for 25-35 cycles (see also Fig. 10).

A. Initial preparation

The two necessary primers, eachcomplementary to the regions flanking theDNA segment to be amplified, areacquired, i.e. bought or synthesized.

DNA is purified from the sample to beanalysed. The starting material may be ablood sample, cells from the buccal mucosa(obtained by a simple mouth wash or bylight scraping with a spatula), blood orsemen stains or hair. (Under suitableconditions it is possible to use the PCRtechnique on non-purified DNA, e.g. froma droplet of whole blood.)

PCR

Figure 10.PCR amplification of DNA

The reaction mixture is heated to90-95 ˚C. This denatures the targetDNA (makes it single-stranded).1

The temperature is lowered to50-60 ˚C. The primers base-pair with complementarysequences in the target DNA.

2The temperature is raised to ca.72 ˚C. This initiates thesynthesis, by DNA polymerase, ofnew DNA strands starting fromthe 3’ position of the primersusing the single-stranded targetDNA as template.

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B. Preparation of the reaction mixture.

To a suitable amount of the DNA samplethe following is added: the two primers, thefour deoxyribonucleoside triphosphates(dATP, dCTP, dGTP, and dTTP), reactionbuffer, and DNA polymerase.

C. Running the reaction - the PCR machine.

The PCR apparatus is programmed for asuitable number of cycles at the desiredtemperatures in order to accomplishdenaturation of the DNA, binding of theprimers, and synthesis of new DNA strandsrespectively.

The reaction is started by initiating theprogramme. A single cycle of the threetemperature steps normally takes three to

four minutes. Each cycle will, theoretically,double the quantity of target DNA.

Analysis of the amplified DNA.The sizes of the STR alleles are determinedby electrophoresis of the PCR products.Sophisticated computer controlled laserequipment is now used to analyse themigration of the PCR products andcompare them with DNA molecules ofknown size that co-migrate with the PCRproducts on the gel. Figure 11 shows acomputer printout from such an analysis.

Problem No. 4:Discuss the results of the analysis shown inFigure 11 and give your opinion about theexpected court ruling based on these results.

Figure 11. Results from a PCR-based DNA profiling in a case of disputed paternity

The two loci analysed: HUMvWA (peaks 16, 17, 18, 19, 20) and HUMF13 (peaks 3.2, 5, 6) both have a repeatedsequence of four base pairs. Each peak represents an allele of the locus in question, the number indicates thenumber of repeats. Primers are chosen to create PCR products of distinct sizes so that several loci can beanalysed in the same system without overlap. This explains why the smaller HUMF13 alleles (3 to 6 repeats) inthe present analysis are represented by larger PCR products than the HUMvWA alleles (16 to 20 repeats). Peak3.2 is a common allele (10 % frequency) that has three repeats and an extra two base pairs.

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Pros and cons of PCRThe PCR technique has revolutionizedDNA analysis and PCR-based DNAprofiling is about to replace RFLP analyses.Modern DNA profiling is both faster andless labour intensive as many of the stepscan be automated. It is possible in a singlereaction to amplify DNA from 4-6 STRregions.

The PCR technique also makes it possibleto carry out analyses on samples containingvery little DNA. Classical DNA profilingneeds at least 20 nanograms of intactDNA, while one nanogram is sufficient fora routine PCR-based DNA analysis,although a single cell (less than 10picograms) can be enough (9). Thisdevelopment has therefore been particularlyimportant in criminal investigations wheresamples may be very small and so degradedthat it is only possible to analyse STR lociand only after in vitro amplification. If onlyvery little DNA is recovered, e.g. from veryfew cells to begin with, the amplification ofsome alleles may fail. This is called alleledropout (9).

Traces of unknown substances in extractsfrom stains can inhibit the DNA

The invention and development of the PCR techniqueThe first international paper describing the PCR technique was published in 1985 (6), but the methodonly became widely used in 1988 after the discovery and purification of a thermostable DNA polymeraseand the automation of the process (7). PCR is now carried out on a computer-controlled thermoblock.Many ‘PCR machines’ are now commercially available. A number of different thermostable DNApolymerases, some genetically modified, are also available. The first of them, and still the most commonlyused one, is the Taq polymerase named after the bacterium Thermus aquaticus in which it was found andwhich lives in hot springs. These enzymes tolerate repeated heating to over 90 ˚C thus makingsupplementary additions of enzyme to the reaction unnecessary. When PCR was first used in 1985 itwas necessary to add DNA polymerase after each cycle because the only enzyme available was athermosensitive enzyme from E. coli.

The PCR technique was invented by Kary Mullis in 1983. He had the idea while driving to his summerhouse in the mountains of California (8). Mullis got a bonus of 10,000 US$ from the company heworked for. Later the company sold the patent right for 300 million US$. Kary Mullis was awarded the1993 Nobel prize in chemistry.

polymerase thus hampering the PCRamplification. In criminal cases purificationof the DNA is often a tedious challengebefore a successful PCR amplification canbe obtained.

The high sensitivity of PCR is also itsweakness; contamination with DNA fromother individuals may give rise to falseresults and wrong conclusions. Laboratoriesworking with PCR amplification from smallamounts of material have therefore had todevelop demanding guidelines for thehandling of samples. The risk ofcontamination of samples with foreignDNA, e.g. from the personnel handlingthem, must be minimal. Amplified DNAfrom other samples is another obvioussource of contamination. Therefore, inforensic laboratories there is a clear-cutphysical separation between areas whereDNA is extracted and purified and areas inwhich the amplification and the subsequentanalysis is carried out. Furthermore, muchattention is paid to the destruction ofamplified DNA after analysis and tolimiting the possibility of amplified DNAescaping from the laboratory.


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Application in practiceFor obvious reasons forensic laboratoriestry to agree on which VNTR regions theywill analyse. Before deciding, aninvestigation is carried out in order toclarify which alleles are present, and atwhich frequencies, in the population inquestion. This information is essential forthe evaluation of the usefulness of the locifor DNA profiling, either in criminal casesor for the elucidation of a disputedpaternity. Figs. 8 and 9 show examples ofthe allele distribution in two VNTR regionsamong Europeans. The mutation rate ofeach locus of interest is also analysed andconsidered. This is essential knowledgewhen using DNA profiling in cases ofdisputed paternity or establishing otherkinds of family relationships.

Forensic genetics in DenmarkIn Denmark all genetic analyses for thejudicial system are carried out by theDepartment of Forensic Genetics, Instituteof Forensic Medicine, at the University ofCopenhagen. Until 1990 only results fromtraditional genetic markers such as bloodgroups (ABO, Rhesus, and MN), tissueantigens (HLA) and various blood enzymesand other proteins were accepted in thecourts.

During the latter half of the 1980’s RFLP-based DNA profiling was developed andused for the first time in court in 1990;initially as a supplement to traditionalanalyses. Today (late 1997), the traditionalmarkers have been practically abandoned incriminal case work. In paternity andimmigration cases DNA profiling hassubstituted tissue typing whenever extendedgenetic analyses are needed.

PCR-based DNA profiling became routinein paternity and immigration cases in early1995. A single STR analysis replaced theRFLP-based analyses of the five largerVNTR regions used previously. The sameSTR analysis has also been used in criminalcases since the middle of 1996, as a

supplement to the RFLP-based analyses.More recently DNA profiling based onamplification and analysis, in one singleprocedure, of four more STR loci has beendeveloped. This ‘package’ is now usedroutinely in paternity and immigration casesand is planned for routine use in criminalcases from January 1998.

There is no doubt that PCR-based DNAprofiling will soon have substituted theRFLP-based analyses in Europe as in theUSA. This is both for practical reasons (seePros and Cons of PCR, p.18) and because theability to discriminate between DNAprofiles is as good or better using PCR-based analyses.

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Criminal casesIn violent crimes (murder, rape) thechallenge is to find out who is the source ofa given stain, or other biological remainssuch as hair, found on the victim or at thescene of the crime. If the DNA profile ofthe stain is different from that of a suspect,the latter can normally be excluded frombeing the source. However, if the profilematches one cannot automatically concludethat the suspect is, in fact, the perpetrator.The possibility of more than one personfitting a given DNA profile cannot becompletely excluded. As mentioned earlieridentical twins must have identical DNAprofiles, but sibs in general are also likely tohave identical profiles if only a few loci areanalysed. The probability of identical allelesamong sibs is at least 25 % for each locus.If four loci are analysed the probability ofidentical profiles among two sibs is at least0.4 % (the minimum values are for the mostfrequent situations where both parents areheterozygous and do not share alleles). Thevalue of DNA profiling may therefore besomewhat limited if two closely relatedpersons, e.g. two brothers, are both suspectsin a case of murder or rape - or both areinvolved in a case of disputed paternity.However, by including more loci thelikelihood of finding distinctive DNAprofiles in non-monozygous sibs keepsincreasing.

Problem no. 5:1. Explain why for a given VNTR locus

the probability of two sibs havingidentical alleles is 25 % or more.

2. Explain why the probability of findingidentical DNA profiles in two sibs is atleast 0.4 % if four VNTR loci areanalysed.

The critical question in a typical criminalcase is the following: If the DNA profile ofthe suspect matches that of the stain, what is theprobability that the suspect is the source of thestain?

The weight which can be attributed to amatch between two DNA profiles dependson how likely it is for a person, picked atrandom from the population, to show thesame profile. In order to estimate this it isnecessary to know the frequencies of thealleles in the population. The expectedfrequency of a specific DNA profile canthen be calculated. A large number ofindividuals have therefore been analysed invarious countries in order to acquireinformation about allele frequencies. If theDNA profile is based on the four mostfrequently analysed RFLP-based VNTRloci, the probability that a person picked atrandom from the population would havethe same DNA profile as a stain from acrime scene is somewhere between 1 in100,000 and 1 in 100,000,000 (10).

In reporting a genetic analysis in a criminalcase, where the DNA profiles from a stainand a suspect match, the routine statementwould be: The likelihood that a person picked atrandom from the population has the same DNAprofile as the stain is less than 1 in 100,000provided that this individual is not closely related tothe perpetrator.

STR loci have fewer alleles than the classicalVNTR loci. It is therefore necessary toanalyse more STR regions in order toachieve the same low probability of arandom match of DNA profiles . Atpresent five STR loci are analysed routinely,four of them in a single PCR amplificationand subsequent gel electrophoresis. Atechnique for analysing six loci in onereaction is currently under development.

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Kinship analysisFor cases of disputed paternity, the task isto find a match for the alleles of the childthat do not match those of the mother. It isobvious that, since each person has twoalleles for every autosomal locus, manydifferent DNA profiles may sharecombinations of alleles.

In short, calculations for classical RFLP-based DNA profiles show that theprobability of a randomly picked malehaving a DNA profile with the necessarycombination of alleles consistent withbeing a certain child’s father is much below1 in 10,000 in most cases. This means thatthe likelihood of picking, by chance, a manwith a DNA profile compatible withfatherhood, and yet who is not the father, isless than 1 in 10,000 - again with the usualreservations for males closely related to thefather.

Thus, in the case of a man with a DNAprofile that does not exclude him frompossible paternity, the conclusion from theanalysis argues in favour of fatherhood witha weight higher than 10,000 to 1. Or, interms of percentages, the likelihood thatthe man in question is the father is higherthan 99.99 % (10).

Resolution of family relationships inimmigration cases may be morecomplicated, depending on the problem tobe addressed and on practical matters suchas access to blood samples from key people.

Mutations as a source of errorVNTR alleles, just as all other DNA,undergo mutations occasionally. This has tobe considered in the evaluation of theresults from a kinship analysis. Somaticmutations, i.e. mutations in ordinary bodycells, will not influence the DNA profile asthey only affect a negligible part of theanalysed DNA (with the exception ofmitochondrial DNA, see later).

Occasionally VNTR regions show a change

in the number of repeats in an allele fromone generation to the next. This can takeplace during DNA replication in theprecursors of the gametes and by so-calledunequal crossing-over during meiosis. Insuch a case a child may inherit an allele notpresent in either parent (see Fig. 11).

VNTR loci used for kinship analyses arechosen from the more stable ones, butmutation rates of 0.1-0.5 % are common,i.e. 1 of every 1,000-200 gametes shows amutation in the VNTR region in question.Consequently it is not possible to exclude aman from a paternity case with a 100 %certainty on the grounds of a mismatch in asingle one of the regions analysed in theDNA profile.

In such cases the situation can almostalways be clarified by including the analysisof three more VNTR loci. Calculationsshow that the probability of an unrelatedman being incompatible in at least onemore of the supplementary three loci isvery high (99.9 %). The chance of twomutations occurring in two out of eight lociis less than 1 in 100,000 (10).

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STR analyses and ethnic groupsHUMTH01 (human tyrosine hydrogenase) isone of the STR loci analysed in PCR-basedDNA profiling. It is located on the short armof chromosome 11 in the first intron (hencethe 01) of the gene encoding the enzymetyrosine hydrogenase. The repeated sequencecontains four base pairs and so far six alleleshave been found among unrelated Danes andfive among a group of unrelated GreenlandEskimos. The frequencies of the differentalleles are very different in the two populations.

Table 1 shows the different patterns of alleledistribution in several populations. Allelefrequencies are important to know becausethey are the basis for the calculations which, ina given criminal case or kinship analysis, try toanswer the critical question: What is thelikelihood that a person with a matching DNA profilehas nothing to do with the case in question, but justhappens to have the same DNA profile by chance?

Problem no. 6:1. Depict the information in Table 1 as

histograms, cf. Figs. 8 and 9.2. Describe the similarities and differences

of allele distribution among thesepopulations.

3. Evaluate the validity of concluding that agiven suspect is the perpetrator (or father)if there is a complete HUMTH01 match ina) a murder caseb) a case of disputed paternity. (Assumethat analyses of other loci have not yetbeen made.)

4. If a justice system based its decisions onsuch a match (question 3), consider thesignificance for different populations.

Analysis of mitochondrial DNAThe use of mitochondrial DNA (mtDNA)analyses is fast gaining ground; both by thejudicial system, for identification, and inanthropological and archaeologicalinvestigations. Its value is partly due to thewide sequence variation found in mtDNA.This makes it highly probable that twoindividuals, not related through links of anunbroken female line, will have one or moredifferences in their mtDNA sequence.About half of the variation is locatedwithin two highly variable non-codingregions encompassing only about 750 basepairs. These are, therefore, the regions ofinterest in this kind of mtDNA analysis.

The mutation rate is higher in mtDNA thanin nuclear DNA. Nevertheless it is fairlyrare to find more than one mtDNAsequence in an individual. However it hasnow been shown, from the large amount ofdata collected, that certain base pairs inmtDNA mutate so often that finding, inone individual, mtDNA with an ‘old’ basepair coexisting with mutated mtDNA is notextremely rare. The mtDNA sequence inone tissue, such as hair, may thus bedifferent from the mtDNA of blood fromthe same individual, in one of these highlymutable base pairs.

Consider the following situation. Hairsfound at a scene of crime have beenanalysed for mtDNA. Comparison with themtDNA of a suspect’s blood sample showsa discrepancy in one base pair, known to beone that frequently shows mutations. Thisis not now considered sufficient evidence toexclude the suspect from the investigation.

Table 1. The distribution of HUMTH01 alleles in different populations.

Allele number (this is the number of repeats)5 6 7 8 9 10 11

Danes 24.6 20.1 12.4 9.5 0.3

Greenland Eskimos 10.9 68.7 5.1 3.7

White Americans 0.5 22.6 15.9 11.0 14.3 0.5

Black Americans 13.5 37.0 21.1 14.6

Hispanic Americans 20.8 33.3 6.8 14.3

Asians 10.4 26.0 5.2 44.2 4.6 0.7


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The most famous example of the presence oftwo different mtDNA sequences in the sameindividual was found during the identification ofthe skeletal remains of the last Russian tsar andhis family, adramatic story initself.

Together with hiswife and their fivechildren tsar NicholasII was executed by theBolsheviks in 1918.What happened to thebodies was kept secretfor many years but in 1979 some peoplesucceeded in locating the grave in which the tsar,his family and members of the household hadprobably been buried. However, it was not untilthe dissolution of the Soviet Union that accesswas given for forensic and anthropologicalanalyses which resulted in the identification offive of the nine skeletons in the grave as beingthose of the tsar, his wife and three of theirdaughters (2,3).

Initially sex determination and STR analyses ofDNA extracted from the bones established thatfive of the skeletons, two from a middle-agedcouple and three from young adult females, werelikely to be from members of single family.Analyses of mtDNA showed that the fourfemales had identical sequences, further evidencethat these were the remains of a woman and herthree daughters. The fact that the woman’s teethwere very well kept with fillings of platinumadded to the evidence that she had been the wifeof the tsar.

The final identification of the middle-agedcouple as being the tsar and his wife was madesubsequently by comparing their mtDNAsequences with those of living persons who,from the available detailed pedigree information,were related to the tsar and his wife, respectively,through unbroken female lineages. HRH PrincePhilip was the key person leading to the

identification of the tsar’s wife. As Prince Philipis the son of a daughter of one of the tsarina’ssisters he is related to the tsarina through an

unbroken femalelineage. The mtDNAsequence of the fourfemales in the graveshould therefore beidentical to his, ifthey were indeed thetsarina and three ofher daughters.It was.

As for the tsar, two living individuals were trackeddown who were related to him through unbrokenfemale lineages. Their mtDNA sequences wereidentical, as expected, and also matched thesequence found in the alleged tsar skeleton.However, a fraction of the mtDNA from theskeleton had a different base pair in one of thepositions. To make a long story short: afterfinding two mtDNA sequences in the extractfrom the skeleton of the presumed tsar it wasdecided to seek permission to analyse mtDNAfrom the skeleton of the tsar’s older brotherGeorgij Romanov who died in 1899 and wasburied in St. Petersburg. His mtDNA showed thesame two sequences which strongly supportedthe authenticity of the tsar skeleton (3).

Finally, in the wake of the elucidation of the tsarstory another ‘mystery’ found its solution. Fordecades a woman had claimed to be Anastasia,the youngest of the tsar’s daughters, who hadallegedly escaped execution in 1918. She died inthe USA in the 1980’s. A preserved sample fromher small bowel was located at a hospital in whichshe had undergone surgery in 1979 and DNAwas extracted and analysed. The mtDNAsequence turned out to be quite different fromthat of the tsarina so the claimed relationshipcould be ruled out. The woman’s identity was finallyestablished when it was shown that her mtDNAsequence was identical to that of a living Polish malewhose maternal grandmother had been, byhistorical evidence, the impostor’s sister (4).

Tsar Nicholas II and his family - a mystery solved

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The quality of samplesDNA from corpses, blood stains and otherbiological remains can be more or lessdegraded and sparse. What kind ofproblems does that bring for the analysisand interpretation of results?

Errors in taking and handlingsamplesHuman beings collect, register and store thesamples and whenever humans are involvedthere is a possibility of error. There areeven examples of deliberate swapping ofsamples. How can these problems beaddressed?

DNA analysis as the onlyevidenceWhat would your verdict be if the result ofDNA profiling was the only evidence ofguilt?

PrivacyDNA analyses of families occasionallyreveal that the social parents are not thebiological ones. It has been estimated that afew percent of children in western societyhave a biological father who is not theperson they think is their father. Somecultures have a broader understanding offamily ties.

How can one ensure that sensitive data likethis does not become accessible to thewrong people.

Databases of DNA profilesShould it be legal for the police to establisha database containing the DNA profiles ofevery individual analysed (convicted or not)in certain criminal cases? If so, who shouldbe authorised to have access to and use thisinformation?

Independence of the DNAlaboratoriesDNA profiling can be carried out withrelatively inexpensive equipment andmaterials. Should there, in your opinion, bespecial compulsory guidelines forlaboratories that undertake such analyses, orshould the market be open so that theprosecution and defence teams can hirecompeting laboratories to carry out theanalysis?

Ethnic problemsThe distribution of allele frequencies invarious VNTR loci can vary amongdifferent ethnic groups. How should onedeal with a situation in which the policedecides to investigate a certain ethnic groupbecause the stain found on a victim has aDNA profile which is more common inthat ethnic group?

SurveillanceIt has been suggested that DNA profilesshould be printed, or encoded, in passportsor social security cards. Discuss both thenegative and the positive aspects of this.Could it be compared to making a completephotographic survey of the population inorder to be able to publish photographs ofwanted persons?

Scientific evidenceResults of analyses, such as DNA profiling,are often presented in court as scientificevidence. How can it be ensured that thedoubts which are always present concerningthe validity of such results will also beadequately discussed in court?

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References

1. Olaisen B., Stenersen M. and Mevåg B. 1997. Identification by DNA analysis of the victims ofthe August 1996 Spitsbergen civil aircraft disaster. Nature Genetics 15: 402-405.

2. Gill P., Ivanov P.L., Kimpton C. et al. 1994. Identification of the remains of the Romanov familyby DNA analysis. Nature Genetics 6: 130-135.

3. Ivanov P.L., WadhamsM.J., Roby R.K. et al. 1996. Mitochondrial DNA sequence heteroplasmy inthe Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II.Nature Genetics 12: 417-420.

4. Stoneking M., Melton T., Nott J. et al. 1995. Establishing the identity of Anna AndersonManahan. Nature Genetics 9: 9-10.

5. Krings M., Stone A., Schmitz R.W. et al. 1997. Neandertal DNA sequences and the origin ofmodern humans. Cell 90: 19-30.

6. Saiki R.K., Scharf S., Faloona F. et al. 1985. Enzymatic amplification of β-globin genomicsequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354.

7. Saiki R.K., Gelfand D., Stoffel S. et al. 1988. Primer-directed enzymatic amplification of DNAwith a thermostable DNA polymerase. Science 239: 487-491.

8. Mullis K. 1990. The unusual origin of the polymerase chain reaction. Scientific American 262

(April): 56-61.

9. Findlay I., Taylor A., Quircke P., Frazier R. and Urquhart A. 1997. DNA fingerprinting fromsingle cells. Nature 389: 555-556.

10. Eriksen B. and Hansen H.E. 1996. Genetikken i retssalene.Dansk Kemi 5: 8-11.

11. Agesen H. et al. (Ed.) 1991. Experimental Gene Technology. Novo Nordisk.

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There are various methods by which

DNA profiling can be simulated in the

school laboratory. Here is a method for

teachers who are fairly familiar with the

practice of DNA gel electrophoresis.

Multiprobe profile simulationThis method uses four DNA samples, oneof which has supposedly come from thescene of the crime, the other threebelonging to suspects. A suspect’s DNAwill match that recovered from the scene ofthe crime; hence three different types ofDNA are needed, each giving distinctivepatterns (once restricted and run on a gel).

To minimise the expense, this exercise usesreadily available DNA from bacteriophagelambda, and a single bacterial plasmid.Some deception is necessary to provide thethree distinctive restriction patterns. This isachieved by restricting one of the lambdaDNA samples with an additional enzymebefore it is given to students for furtherrestriction with HindIII and analysis.Students should not be made aware of thispre-treatment!

Teachers will be able to devise a suitablescenario (e.g. a description of a suitablecrime) to enliven the classroom activity.

Any suitable DNA gel electrophoresisequipment may be used for this work. TheNational Centre for BiotechnologyEducation supplies low cost kits containingroom temperature stable lambda DNA andrestriction enzymes, together with all thenecessary equipment, chemicals andinstructions for DNA gel electrophoresis.Detailed guidelines are given for treatmentof DNA with restriction enzymes and theseparation of the resulting DNA fragmentsusing gel electrophoresis.

Further details can be obtained from theNCBE website at:

http://www.rdg.ac.uk/NCBEor,

NCBEThe University of Reading,WhiteknightsReading, RG6 6AJ, UK.Phone: (44) 118 987 3743Fax: (44) 118 975 0140.

or,Email: [email protected].

For this DNA profile simulation a suitableplasmid is needed in addition (e.g. pUC18,available from molecular biologicalsuppliers - but expensive).

Aim● To simulate DNA profiling and show

some of the essential proceduresinvolved in DNA restriction analysis.

Advance preparationDNA samples must be rehydrated andmixed with EcoRI or a suitable plasmid, asappropriate, before the practical activitystarts.

OrganisationThis activity takes about 50 minutes tocomplete, including a period of 30 minutesduring which the DNA samples areincubated with the enzyme. If using lowvoltage electrophoresis equipment such asthat supplied by the NCBE, running the geltakes between 6 and 12 hours.

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Equipment and materialsRequired by each student or group ofstudents (quantities and concentrationsare as used in the NCBE protocols).

Three ‘suspect’ DNA samples:● tube A, containing λ DNA solution:

2 µg in 20 µl● tube B, containing λ DNA solution:

2 µg in 20 µl, mixed with EcoRI (10units, dried)

● tube C containing λ DNA solution:2 µg in 20 µl plus a suitable plasmid*(e.g. pUC18 or pBR322): 0.2 µg in 2 µl

A ‘scene of crime’ DNA sample, i.e. oneof the three listed above - the cheapestand simplest would be:● tube D, containing λ DNA solution:

2 µg in 20 µl

Also needed:● 4 tubes, each containing 10 units of

HindIII restriction enzyme● an electrophoresis tank● a 4-well gel comb● 15 micropipette tips (this number

includes a couple of spares)● micropipette or microsyringe● bromophenol blue loading dye with

sucrose (about 10 µl)● TBE buffer solution● 0.8 % agarose gel, made up using TBE

buffer solution, molten● about 10 ml 0.04 % Azure A solution,

made up in 20 % ethanol (a safealternative to ethidium bromide)

● marker pen● power supply or batteries (12–25 volts)● water bath or incubator, set at 37 °C

* any plasmid of 4-6 thousand base pairsis suitable.

ProcedureFollow an established procedure forincubation of the samples with HindIII,separation of the fragments by gelelectrophoresis and staining of the resultinggel to visualise the bands of DNA (see theinstructions with the NCBE DNA kits).

ResultsFigure 12 shows the sort of band pattern tobe expected, the ‘scene of crime’ samplecan be matched with one of the ‘suspect’samples. The pattern can be likened to the‘fingerprint’ method of Alec Jeffreys’original work although it should be stressedthat it is only a simulation. Single probetechniques would produce one or twobands per sample.

Figure 12.A DNA gel after electrophoresis and staining

wells

DNA bands

loading dye

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