CH 5-Molecular Tests For Identity SH-28

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AmericanSocietyofCytopathology
CoreCurriculuminMolecularBiology
Copyright2010AmericanSocietyofCytopathology
Broughttoyouby
AmericanSocietyofCytopathology
CoreCurriculuminMolecularBiology
Chapter5
ApplicationsofMolecularTesting
MolecularTestsforIdentity
StephanieA.Hamilton,EdD,SCT,MB(ASCP)
MDAndersonCancerCenter
Houston,Texas
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Timeline of Identity Testing
1800s
1900
1960
1980s
1985
1990s
Fingerprint
ABObloodgrouptyping
HLAtyping
DNAanalysis(VNTRs)
FirstDNAfingerprint
usedincourtcase
DNAanalysis(STRs)
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Mutations and Polymorphisms

Transmissible(inheritable)changeinDNAsequence
isamutationorpolymorphismwhichmayormay
notproducephenotypicdifferences
Mutation: aDNAsequencechangepresentina
relativelysmallproportionofapopulation
Polymorphism: aDNAsequencechangepresentinat
least1%ofapopulation
Variationamongindividualsofnoncoding(doesnot
codeforaprotein),repetitivesequencesinDNAiskey
toidentitytesting
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Types of Polymorphisms
SingleNucleotidePolymorphisms(SNPs)
Asinglenucleotidedifferenceinagenomic
sequence
Example:Humanleukocyteantigen(HLA)locus.
ThisisahighlypolymorphicregionwhereSNPs
occurfrequently.Variabilitycodesforpeptides
thatestablishselfidentityoftheimmunesystem.
Usedfordeterminingcompatibilityoftransplant
donors.
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LongInterspersedNucleotideSequences
(LINES)
Arehighlyrepeatedsequences
68kbpinlength
ContainRNApolymerasepromoters
Containopenreadingframesrelatedtoreverse
transcriptaseofretroviruses
Morethan500,000LINESingenome
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ShortInterspersedNucleotideSequences
(SINES)
0.3kbpinlength
Morethan1,000,000copiespergenome
IncludeAluelements(namedforhaving
recognitionsitesfortheALuIrestrictionenzyme)
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VariableNumberTandem
Repeat(VNTR)
Alocationingenomewherea
shortnucleotidesequenceis
organizedasatandemrepeat
(clusteredtogetherand
orientedinsamedirection)
Alsoknownasminisatellite
sequences
10to100bpinlength
Variations of VNTR (D1S80)

allele lengths in 6 individuals
http://en.wikipedia.org/wiki/Variable_number_tandem_repeat/ Accessed: 6/14/10

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ShortTandemRepeat(STR)
Occurswhenapatternoftwoormorenucleotides
arerepeated
Repeatedsequencesaredirectlyadjacenttoeach
other
Lengthrangesfrom2to16bp
Alsoknownasmicrosatellites
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STR Nomenclature
InternationalSocietyforForensicGeneticsrecommendednomenclaturefor
STRlociin1997
STRswithingenesaredesignatedaccordingtogenename
Examples:
TH01isinintron1ofhumantyrosinehydroxylasegeneonchromosome11
TPOXisinintron10ofhumanthyroidperoxidasegeneonchromosome2
NongeneassociatedSTRsaredesignatedbytheD#S#system
DstandsforDNA,thefollowingnumberdesignatesthechromosomewhereSTR
islocated
Sreferstoauniquesegment,followedbyanumberregisteredinthe
InternationalGenomeDatabase(GDB)
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RestrictionFragmentLengthPolymorphism(RFLP)
Adifferencebetween2ormoresamplesofhomologousDNA
moleculesarisingfromdifferinglocationsofrestrictionsites
WasthefirstDNAprofilingtechniqueusedforgenemapping,
humanidentification,andparentagetesting
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Summary of Types of Polymorphisms

and Laboratory Methods
Polymorphism Structure
Detection Method
RFLP
One or more nucleotide changes

that affect the size of restriction
enzyme products
Southern blot
VNTR
Repeats of 10-50 base sequences Southern blot, PCR

in tandem
STR
Repeats of 1-10 base sequences

in tandem
PCR
SNP
Alterations of a single nucleotide
Sequencing, other
Buckingham L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis, 2007.
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RFLP Analysis
RFLPanalysis
DNAsampleisbrokenintopieces(digested)byrestriction
enzymes
Resultingrestrictionfragmentsareseparatedaccordingto
lengthsbygelelectrophoresisanddetectedbySouthern
blot
Fragmentsizesmayvaryduetochangesinnucleotide
sequenceinorbetweentherecognitionsitesofa
restrictionenzyme
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RFLP Analysis
AsmallsegmentofgenomeisbeingdetectedbyaDNAprobe
(thickerline)
InalleleA,genomeiscleavedbyrestrictionenzymeatthree
nearbysites(triangles)
Onlyrightmostfragmentwillbedetectedbyprobe
Inallelea,restrictionsite2hasbeenlostbyamutation
Probenowdetectsthelargerfusedfragmentrunningfrom
sites1to3
http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.

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RFLP Analysis
Inthisexample,theprobeandrestrictionenzymearechosen
todetectaregionwhichincludesaVNTRsegment(boxes)
Inallelecthereare5repeatsintheVNTRandprobedetects
alongerfragmentbetweenthe2restrictionsites
Inalleledthereareonly2repeatsintheVNTR,soprobe
detectsashorterfragmentbetweenthesame2restriction
sites
http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.

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RFLP and Parentage Testing
AgeneorregionofDNA(locus)willhave
severalversions(alleles)oftheDNAsequence
withinthatgene
Eachgenecanhavedifferentalleles
DifferentDNAsequences(alleles)canresultin
differenttraits,suchascolor
DifferentDNAsequences(alleles)canhavethe
sameresultintheexpressionofagene
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DNA-based Parentage Testing
Humansarediploid:eachpersonhas2alleles
orcopiesofeachlocus
Homozygous:allelesarethesame
Heterozygous:thetwoallelesaredifferent
Morecloselyrelatedindividualsarelikelyto
sharemoreallelesthanunrelatedpersons
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DNA-based Parentage Testing

DNAisinheritedasonechromosomecomplement
fromeachparent
Eachchromosomecarriesitspolymorphism
Offspringinheritsacombinationoftheparental
polymorphisms
Thus,throughRFLPanalysis,onecaninferaparents
contributionofallelestoasonordaughterfromthe
combinationofallelesinthechildandthoseofthe
otherparent

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Through RFLP analysis, the fragment

sizes of the child (electrophoresis lane 2)
is a combination of those from each parent
(father, lane 1) and (mother, lane 3)
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1
http://en.wikipedia.org/wiki/Polymerase_chain_reaction.
Accessed 6/15/2010.
Electrophoresis of PCR-amplified DNA fragments.

(1) Father. (2) Child. (3) Mother. The child has inherited
some, but not all of the fingerprint of each of its parents,
giving it a new, unique fingerprint.
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Animation
http://www.rvc.ac.uk/review/DNA_1/4_VNTRs.cfm
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In a paternity test, alleles or fragment sizes of
offspring and mother are analyzed.
Remaining fragments (ones that do not match
mother) have to come from the father.
Alleged fathers are identified, or included,
based on the ability to provide remaining
alleles.
Aside from possible mutations, a difference in
just one allele may exclude paternity.
In this figure, only two loci are shown. Of the
alleged fathers (AF) shown, only one could
supply the fragments not supplied by the
mother.
Which is the alleged father?
Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis Company, 2007.
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Parentage Testing
Aparentagetestrequiresanalysisofatleasteight
loci.
Themorelocitested,thehighertheprobabilityof
positiveidentificationofthefather.
PaternityIndex(PI):
Calculatedforeachlocusinwhichallegedfatherandchild
shareanallele
Isanexpressionofhowmanytimesmorelikelythechilds
alleleisinheritedfromallegedfatherthanbyrandom
occurrenceofthealleleinthegeneralpopulation
AlleleoccurringfrequentlyinpopulationhaslowPI;arare
allelehasahighPI
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Other Methods for Detecting

Polymorphisms
IncontrasttoVNTRs,smallerSTRscanbeamplified
byPCR
Lessspecimenneeded
Shortertimeforanalysis
Multiplexingandautomationisfacilitated
Canusefluorescencetechnologyandcapillary
electrophoresis
Reversedotblotprocedure:usesanarrayof
immobilizedprobestodetectsequence
polymorphisms
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Gender Identification
Amelogeningene
LocatedontheXandYchromosomes
NotaSTR
Amelogeninlocus
Polymorphismlocatedinsecondintronofamelogenin
gene
YalleleofgeneissixbplargerinthisregionthanXallele
Amplificationandelectrophoreticresolutionreveals2
bandsorpeaksformales(XY)andonebandorpeakfor
females(XX)
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Amelogenin Locus
Males are heterozygous for the amelogenin locus (XY), and

females are homozygous for this locus (XX). Amplification of
amelogenin will produce a male-specific 218 bp product (Y allele)
in addition to the 212 bp product found on the X chromosome (X
allele).
Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications.
Philadelphia: F.A. Davis Company, 2007.
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Y-STRs
YSTRsarerepresentedonlyoncepergenomeand
onlyinmales
AsetofYSTRallelescomprisesahaplotype,or
seriesoflinkedallelesalwaysinheritedtogether,
becauseYchromosomecannotexchange
information(recombine)withanotherY
chromosome.
MarkerallelesonYchromosomeareinheritedfrom
generationtogenerationinasingleblock.
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Y-STRs
LesspowerfulthanautosomalSTR
Usefulwhereevidenceconsistsofamixtureof
maleandfemaleDNA(ex.rapecases)
Usedalsoinlineagestudiesinvolving
paternallylinkedrelationshipsand
identification(ex.inheritanceclaims)
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Mitochondrial DNA
Mitochondriacontainacirculargenomeof16,569
basepairs
StructureistwostrandsofcircularmtDNA
HaveanasymmetricdistributionofGsandCs
GeneratingaGrichheavy(H)strandandaCrichlight(L)
strand
mtDNAareinheritedmaternally
Usedinforensicanalysis(ex.opencasefiles,missing
personscases,andtypingancientspecimens)
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Identity Testing: Applications
Applications
Parentage
Immigration
Criminalinvestigation(forensics)
Clinical
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Application: Immigration
TheFirstCase,1985
AyoungboyfromGhanaleavestheU.K.tobereunitedwithhis
fatherinGhana.HethenreturnstotheU.K.Tobereunited
withhismother.
Washethesonorthenephewofthiswoman?
HLAtechniquesatthattimeweretoocrudetoresolvethisissue.
Usingminisatellite probesforhypervariable regionsinhuman
DNA,AlecJeffreys indisputablyshowedthatthiswasindeed
thesonandnotthenephewofthiswoman.Thisimmigration
caseledthewayforDNAfingerprinting.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American Association for Clinical
Chemistry (AACC) Press, 2002.
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Application: Forensics
TheFirstMurderCase,1986
TwoschoolgirlsinthequiettownofLeicesterwere
murdered,onein1983andonein1986.The
circumstancesoftherapesandmurderwere
identical.Sowhenayoungmanconfessed,the
authoritiesthoughttheyhadtheassailantforboth
crimes.Semenswabshadbeentakenfrombothgirls
andDNAanalysiswasrequested.Thesampleswere
fromthesameindividual,butnottheonewhohad
confessed.
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Application: Forensics
TheFirstMurderCase,1986(cont.)
Acallwasthenputouttoalllocalmalesbetweenthe
agesof17and34tobesubjectedtoDNAfingerprint
analysis.Thetruemurdererhadaworkmategoinhis
place.Astheinvestigationdraggedon,theproxytold
afriendatthelocalpubthathehadstoodinfor
someoneaspartoftheDNAtest.Awoman
overheardtheconversationandreportedthisto
authorities.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American Association for
Clinical Chemistry (AACC) Press, 2002.
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Applications for Identity Testing in

Clinical Laboratory
Specimenidentification
Urinedonoridentification(toxicology)
Preemploymentdrugtesting
Confirmationforothercivilcases
Monitoringbonemarrowtransplant
chimerism
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Application: Specimen Identification
A51yearoldmanunderwentcorebiopsyofthe
prostatetoruleoutcarcinoma.SeveralH&E
slideswerereceivedthatshowedafragment
ofcarcinomatousepitheliumadjacentto
benignprostatecoresamples.Theoriginof
theneoplastictissuewasquestioned.
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Specimen Identification Testing Results

Specimen
LDLR
GYPA
HBGG
D7S8
GC
TPO
THO
Blank
-----
-----
-----
-----
-----
-----
-----
Control
B,B
A,B
A,A
A,B
B,B
182/194
243
Prostate
B,B
A,B
A,B
A,A
C,C
230 bp
181 bp
? Tissue
A,B
A,B
A,B
A,A
A,C
230 bp
181/189 bp
The blank and control results indicated there were no problems

during the testing; however, since there are 2 loci (LDLR and GC)
that differ from the patients benign prostate tissue and the neoplastic
tissue, it is possible there was contamination during the tissue
processing.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC:
American Association for Clinical Chemistry (AACC) Press, 2002.
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Application: Pre-employment Drug

Screening
Apreplacementurinedrugtestconductedina
youngmanwaspositivefor480ng/mL
benzolylecognine.Whencontactedbythe
medicalreviewofficer,thedonordeniedusing
drugsandsaidthecollectormusthavemixed
upsamples.
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Pre-employment Drug Screening Results

Specimen
DQA1
LDLR
GYPA
HBGG
D7S8
GC
Blank
-----
-----
-----
-----
-----
-----
Control
1.1,4
B,B
A,B
A,A
A,B
B,B
Urine
1.1,1.2
A,A
A,B
B,B
A,A
B,B
Blood
1.1,1.2
A,A
A,B
B,B
A,A
B,B
The blank and control results indicated there were no

problems during testing; however, a blood sample taken
from the applicant matched the loci of the applicants urine
sample, thus confirming the urine sample was his.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American
Association for Clinical Chemistry (AACC) Press, 2002.
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Bone Marrow Transplant Typing and

Monitoring
Potentialdonorsaretestedforimmunological
compatibility
PerformedbyanalyzingHLAlocususingsequencespecific
PCRorsequencebasedtyping
Engraftment:Donorcellsreconstitutetherecipients
bonemarrow
Recipientisgeneticchimera(recipienthasbodyand
bloodcellsofseparategeneticorigins)
EngraftmentmonitoringbyPCRamplificationofSTRs,
capillaryelectrophoresis,andfluorescentdetection

CH 5-Molecular Tests For Identity SH-28

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Timeline of Identity Testing

Mutations and Polymorphisms

Variations of VNTR (D1S80)

http://en.wikipedia.org/wiki/Variable_number_tandem_repeat/ Accessed: 6/14/10

Summary of Types of Polymorphisms

One or more nucleotide changes

Repeats of 10-50 base sequences Southern blot, PCR

Repeats of 1-10 base sequences

Alterations of a single nucleotide

http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.

http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.

RFLP and Parentage Testing

DNA-based Parentage Testing

DNA-based Parentage Testing

RFLP and Parentage Testing

Through RFLP analysis, the fragment

Electrophoresis of PCR-amplified DNA fragments.

RFLP and Parentage Testing

RFLP and Parentage Testing

Which is the alleged father?

Other Methods for Detecting

Males are heterozygous for the amelogenin locus (XY), and

Identity Testing: Applications

Applications for Identity Testing in

Application: Specimen Identification

Specimen Identification Testing Results

The blank and control results indicated there were no problems

Application: Pre-employment Drug

Pre-employment Drug Screening Results

The blank and control results indicated there were no

Bone Marrow Transplant Typing and

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