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EBookCrimeSceneDnaFingerPrinting

DNAFingerPrinting
WrittenbyDr.D.Rao

FINGERPRINTING:

DNAfingerprinting(DNAtyping,DNAidentification,orgenetictyping)isatechniqueinvolvingchemicallydividingtheDNAintofragmentswhichformauniquepatternand
then matching that identity profile with the pattern obtained from similarly testing a suspects blood specimen. If the two patterns match, the possibility of error, i.e. the
chancethattheydonotbelongtothesameindividualmaybelessthanonein30billion.Dr.AlecJeffreysin1985,developedDNAfingerprinting.
Human body consists of about six thousand billion cells which constitute tissue and organ systems. Every living cell has genetic material contained in units called
chromosomes,whicharelocatedinnucleus.DNAispresentonlyinnucleatedcells.Eachhumansomaticcellhas23pairsofchromosomesofwhich23arederivedfrom
biological father, and 23 from the mother, due to fertilization of ovum with sperm. Genes are arranged along the length of each chromosome, which are responsible for
variousfunctionsofthebody.Eachgenecarriesinstructionsfortheproductionofaparticularproteinwhichperformsaparticularfunction.Genesarealsoresponsiblefor
transmissionofheredity.Thegenesaremadeupofchemicalmoleculescalleddeoxyribonucleicacid(DNA).Thehumangenomecontainsabout6x10DNAmoleculesper
diploidgenome.
The core of the chromosome is a very long and extremely thin thread of DNA. A single human chromosome is about 1/12,500cm. long. The DNA molecule in this
chromosomeisabout2.5cm.inlength,compactedintothechromosomebysuccessivecoiling.
The total DNA in a cell is about 180 cm. in length. Each chromosome consists of two long linear DNA molecules, the polymers being hydrogen bonded via specific
nucleotide pairing and coiled as a double helix which is spiral in nature, and looks like a spiral staircase. The helix is structurally stabilized by nuclear proteins called
histones being referred to as chromatin. Chromatin may be condensed to varying degrees of compactness and in its most compact form is seen microscopically as
chromosomes at the metaphase stage of each cell cycle. The chromosomes are continuous strands of DNA ranging from 50 to 500 million lolecules per chromosome,
encodedinthis.
Eachnucleotideiscomposedofphosphate,deoxyribosesugar,andorganicnitrogenousbase.Thebasesareadenine(A),guanine(G),cystosine(C),andthymine(T).The
bases of one strand are connected to the bases of the other strand b hydrogen bonds, while adjacent nucleotides are linked with each other by covalent bonds. Adenine
combinesonlywiththymineadguaninecombinesonlywithcytosine.TheDNAmoleculeresemblesatwistedropeladderwithfourkindsofstairsteps,e.g.,AT,TA,CG,
orGC.therearethreehydrogenbondsbetweenGandC,and2bondsbetweenAandT.
A single DNA molecule consists of 50 to 500 million base pairs. The two strands of DNA helix run in opposite direction. The base sequence of one strand is always
complementarytothesequenceontheother.EachsegmentofDNAinachromosomewhichcodesforaparticularproteiniscalledagene.Inthehumangenomethereare
about 10,000 genes, accounting for about 5% of the entire cellular DNA. In between the active base pairs which code fro a particular protein, there are large number of
redundant/inactivebasepairsforming95%ofDNA,whichisconsideredasjunkDNA.InjunkDNAshortsequencesofbase,repeatthemselvesoveragainlikeastutter
(repetitive DNA), e.g. GCTA, GCTA, GATA, GATA, etc. The regions containing repetitive DNA demonstrating, hypervariability from person to person are called satellite
DNA,whichshowsanextremelyhighdegreeofvariability,andthesevariantsarecalledvariablenumbertandemrepeats(VNTR)orminisatellites.therearemorethan
1500 VNTRs in the human genome. Selected regions of VNTR are broken into fragments using special enzymes (restriction endonucleases), which are individualiatic in
natureandestablish100%identity.
TherearetwomethodsofDNAanalysisincommonuse.(1)RELP(restrictionfragmentlengthpolymorphism).(2)PCR(polymerasechainreaction).
(1)RELPMethod:DNAcanbeextractedfromanybodyfluidortissueinwhichnucleatedcellsarepresent.Allthesamplesshouldbefrozensemen,hairroots,toothpulp,
tissuefromanyorgan,orskin)areusuallyexamined.TheseparationofDNAinvolves.(1)distruptionofcellsandfractionationofcellularorganelles,(2)dissociationofDNA
fromproteinsbytheuseofsaltsolutionordetergent,(3)additionofanextractanttophaseseparatethebulkoftheproteinfromtheDNA,(4)useofenzymesordifferential

precipitation to remove the RNA and polysaccharides. The isoloated DNA is quantitated by ultraviolet spectrophotometry DNA is completely digested with restriction
enzymescalledrestrictionendonucleases.TheseenzymesrecognizethespecificsequenceinthedoublestrandDNAandcuttheDNAatthissiteintovariousfragments,
calledasrestrictionfragmentlengthpolymorphism(RFLP).RFLPsareproducedduetovariationsinhumanDNA.Thesevariationsinrestrictionfragmentlengthsisdueto
presence of variable number of tandem repeats (VNTR). Most of repeated DNA are arranged as short sequences repeatedly contiguously in tandem, hence called VNTR.
Several restriction rndonucleases are in use, e.g., EcoRI, PsT1, HinFI (obtained from E.coli), sau 3A1 (obtained from Staphylococcus aureus), HaeIII (obtained from
Haemophilusinfluenzae).PsT1(asixbasecutter)iscommonlyused,whichrecognizesthesequenceCTGCAG.
Digested DNA is run on a agarose gel electrophoresis. The different restriction fragments are separated varying in length between 0.5 to 25 kb, which varies from one
individual to another. The smaller fragments move much faster through the gel than the larger one. The gel is later stained with ethedium bromide for 40 minutes which
tightlybindstoDNAandfluorescesunderUVlight.
From the agarose gel, DNA is transferred to nylon membrane using capillary transfer technique of Southern. The result is a mirrorimage replica of fragment distribution.
Vacuumblottingoftransferisusedmorecommonlyasitislesstimeconsuming.DNAisthenfixedbyheatat800CorcrosslinkedbythecationofUVirradiation.
Next hybridization is done which is the pairing of two complementary single strands of DNA to form double stranded DNA. It involves the addition of a probe to the nylon
membrane. A probe is a singlestranded recombinant DNA. Segment, or synthetic DNA, which is designed to go to a particular predetermined locus on a particular
shromosome.Itisusuallytaggedwitharadioactivemarker,suchasP.TheprobescansalltheDNAfragmentsandwhereveritencountersitscomplementarysequence,it
willhybridisewithit,therebymakingthefragmentradioactive.Usuallyfourprobesareusedoneatatime,duetowhichfourdifferentregionsoftheDNAwouldbealalysed.
IntheCentreforDNAFingerprintingandDiagnostics(CDFD),Nacharam,Hyderabad76,A.P.India,Bkmprobeisused,whichisamultilocusprobeisolatedfromthefemale
bandedkrait,asaminorsatelliteDNA.Morthan36hypervariableVNTRaredetectedbyBKm.
Thelabelincorporatedintotheprobepermitstolocalizethesesites.Afterhybridization,themembranesarewashedwith0.05%SDSwhichremoveslooselyboundprobe.
themembraneisthenwrappedinthesaranwrapandplacedintheXraycassetteholderalongwithXrayfilm,andkeptatminus80C.Exposuretimedependsuponthe
specificactivityoftheproberangingfromfewhourstodays(upto10days).Xrayfilmsarethendevelopedandfixedintherespectivereagentsandfinallywashedinwater
and dried. This autoradiograph is a permanent record, in which grey to black bands are seen where the radioactive probes had hybridized to fragments bearing
complementarysequences.Theseriesofbandsseenonthefilmiscalledanautorod,whichrepresentstheDNAfingerprintofthatindividualfromwhomtheDNAhadbeen
obtained.Thepatternofbandsisuniquetoeachindividual.
DNAFINGERPRINTING:Allnucleatedcellsinthebodycontain23pairsofchromosomes.EachchromosomeconsistsofadoublespiralofDNAintheshapeofatwisted
ladder,thedoublehelix.TheDNAiscomposedoftwostrandsofsugarandphosphatemoleculesthataretwistedintoadoublehelixthatisboundtogetherbylinksformed
from adenine and thymine, and cytosine and guanine. Each helix twist has ten such links, like rungs on a twisted ladder. A single DNA molecule has thousands of such
links, and the permutations of the bases in each adjacent link create the genes. The genes are separated by segments having no genetic function. Each human nucleus
containsaboutametreofDNA,butonly10%isusedforgeneticcoding,therestbeingredundantorsilentsegments(stuttershypervariableregions(HVR)ministatellites).
Of these redundant segment, there may be 200 to 14,000 repeats of each identical sequence on each DNA strand. These segments of nucleotides are called repetitive
DNA. The length, constitution, and number of the repetitive sequences are different for each person, but are unique for an individual (except uniovular twins), and are
transmittedfromparentsinregularfashion.Thismethodisasuniqueasfingerprintstoanindividual.
NucleatedcellsarethesourceofDNAforextractionfromblood,semen,vaginalepithelialcells,toothpulp,bonemarrow,hairroots,muscle,skin,mucousmembranes,etc.
AsampleistakenandfromittheDNAischemicallyextractedandpurified.Itisthencutintofragmentsatspecificbasesequencesbyarestrictionenzyme(RE).Whenthis
process is repeated by several enzymes, each of which cuts at different sites, enough information is gathered to construct a detailed genetic fingerprint of a person.
BecauseeverypersonsDNAspecimenfromoneindividualtoanotheraredifferentinnumberandlength,fromthoseinaDNAspecimenfromanotherindividual.Next,the
double stranded DNA are denatured into single strands. The DNA fragments are then separated by gel electrophoresis a negative charge and thus travels towards the
positivepoleinelctrophoresis.Thedistancethateachfragmenttravelsdependsonitslength.Thelongerthefragment,thesloweritsrateofmigration.AnumberofDNA
bandsareproducedinthegel.ThesearetransferredtonylonornitrocellulosesheetsbySouthernblottingtechnique.ThemembraneisthenexposedtoaDNAprobe,which
is a single strand of DNA labeled with radioactive phosphorus 32, which specifically binds to the core sequences. Xray film is put in direct contact with probelabelled
membranetodetecttheradioactivepatterns,whichappearasaseriesof30to40parallelbandsorbars.EachdarkbandrepresentsapointwheretheDNAprobeisbound
to its complimentary base sequences. From the presence of different bars in given positions, comparisons can be made with other samples. It is possible to identify a
personfromasinglehumancell.Thechanceoftwopersonssharingthesamesequenceisaboutoneinthirtythousandbillions.Rarely,therearemutationsconfinedtoone
band.

DNAanalysis
1.Frombodyofthediseased.
Thebiologicalsamplesshouldbecollectedinfollowingpriorityorderincaseofdeadperson.
a. Skeletalmuscle(deepmuscle)/TissueTheconcernedM.O.shouldcollecttheleastdecomposedportionofthetissueanditassoonaspossibletoavoidfurther
decomposition.
b. ToughtissueIfthedecompositionhasalreadystartedcollectthetougher
tissueforwhichthedecompositionrateiscomparativelyslow.
Followingtypesoftissueareobservedtohavelesserdegreeofdecomposition.
1. Muscletendons
2. Footi.e.heelskin
3. Scalpskin

4. Palmskin
5. Stomachwall
c. ToothForwordalltheteethpresentwiththebody.
d. ScalphairwithrootsForwordbunchofscalphairwithroots.Pluckthescalphairdontcutthemwithscissors.
e. P.M.Blood.
f. BonesIftheskeletonisationofthedeceasediscompleteandnotissueorotheroptionsareavailable,thenthelongerbonesofthebodysuchasFemurshouldbe
forworded.Ifsomedriedtissueortendonsarestuckupwiththebones,thendonotremoveit,astherearesomechancestogettheDNAfromit.

2.CrimesceneandotherExhibits
1. Blood stains In murder, attempt to murder and other related cases, blood stained clothes, scrapings, blood stained weapons and other related exhibits should be
forwardedaspertherequirement.

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TOP

2. SemenstainsInrapecases,clothesofvictimandaccused,clothesatcrimescene,condomsandotherrelatedexhibitsshouldbeforwarded.
Modeofparcelandproperpreservativesforrespectivesamples.

Sr.

Sample

Modeofparcel

Preservative

Tissue,Muscle

Putthesampleinclean,sterileplasticor

DMSO or Normal physiological

piecescalpskinetc.

glasscontaineraddthepreservativeasrecommended.Bringthesampleinice.

No.
01

saline or 4% EDTA solution or

keepthetissueasitisin20 0
Crefrigerator.
02

Blood

CleansterileglassvialforP.M.blood.Forcontrolbloodsamples,tubesaresuppliedbyFSL. 4%EDTA
Bringthesamplesinice.

03

Teeth

Airdryalltheteethavailable,putthemindry,cleanandsterileplasticorglasscontainerand Nopreservative
forward.

04

Scalphair

Airdrythesample,putindry,cleanandsterileplasticorglasscontainerandforward.

Nopreservative

05

Bone

Air dry and wrap in clean brown paper. Do not macerate or treat with any chemical. If the Nopreservative
tissueortendonisstuckuptothebone,keepasitis,donotseparateordisturbit.

06

Blood stained clothes Air dry the clothes and wrap in clean brown paper. Dont pack clothes in wet or semi wet Nopreservative
andscrapings

condition. In case of blood stains on the wall, scrap with a clean new razor blade. While
scraping, take the precaution that paint on the wall does not mix with the blood. Put the
scrapingsoncleanpieceofpaperandpreparethepacket.

07

Semenstains

Air dry the clothes and wrap in clean brown paper. Do not pack clothes in wet condition. In Nopreservativ
case of used condom, reverse it carefully to bring its inside out, empty its contents on a
clean dry & sterile piece of cloth. Dry the cloth and forward. Also dry the condom pack in a
brownpaperandforward.

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