核苷酸多态性4

MultiplexPCRandminisequencingofSNPs—

amodelwith35YchromosomeSNPs

JuanJ.Sancheza,*,ClausBørstinga,CharlotteHallenberga,AndersBucharda,

AlexisHernandezb,NielsMorlingaDepartmentofForensicGenetics,InstituteofForensicMedicine,UniversityofCopenhagen,11FrederikV’sVej,

DK-2100Copenhagen,Denmark

b´a,CampusdeCienciasdelaSalud,38320LaLaguna,Tenerife,SpainDepartamentodeCanarias,InstitutoNacionaldeToxicologı

Received22January2003;receivedinrevisedform2July2003;accepted7July2003

aAbstract

Wehavedevelopedarobustsinglenucleotidepolymorphism(SNPs)typingassaywithco-amplificationof25DNA-fragments

andthedetectionof35humanYchromosomeSNPs.ThesizesofthePCRproductsrangedfrom79to186basepairs.PCRprimersweredesignedtohaveatheoreticalTmof60Æ58Catasaltconcentrationof180mM.Thesizesoftheprimersrangedfrom19to34nucleotides.TheconcentrationofamplificationprimerswasadjustedtoobtainbalancedamountsofPCRproductsin8mMMgCl2.Forroutinepurposes,1ngofgenomicDNAwasamplifiedandthelowerlimitwasapproximately100pgDNA.Theminisequencingreactionswereperformedsimultaneouslyforall35SNPswithfluorescentlylabelleddideoxynucleotides.Thesizeoftheminisequencingprimersrangedfrom19to106nucleotides.Theminisequencingreactionswereanalysedbycapillaryelectrophoresisandmulticolourfluorescencedetection.FemaleDNAdidnotinfluencetheresultsofYchromosomeSNPtypingwhenaddedinconcentrationsmorethan300timestheconcentrationsofmaleDNA.Thefrequenciesofthe35SNPsweredeterminedin194maleDanes.ThegenediversityoftheSNPsrangedfrom0.01to0.5.#2003ElsevierIrelandLtd.Allrightsreserved.

Keywords:Ychromosome;Singlenucleotidepolymorphism;MultiplexPCR;Minisequencing;Genotyping

1.Introduction

Alargenumberofsinglenucleotidepolymorphisms(SNPs)havebeenidentified[1].InvestigationsofSNPsontheYchromosomeinvariouspopulationshavegivenusimportantinformationonthehistoryofthehumanmalepopulations(e.g.[2–8]).DuetothelowmutationratesofSNPs,theinformationrelatestolongerperiodsoftimecomparedtotheinformationobtainedwithe.g.shorttandemrepeat(STR)[9–11]andminisatellitemarkersas,forexam-pleMSY1[12,13].

Presently,typingofselectedshorttandemrepeat(STR)systemsisthestateoftheartinforensicroutinecasework.Itis,however,anticipatedthatSNPtypingwillbeusedfor

Correspondingauthor.Tel.:þ45-35-32-62-25;fax:þ45-35-32-61-20.

E-mailaddress:juan.sanchez@forensic.ku.dk(J.J.Sanchez).

*parentagetestingandforensiccaseworkinthefuture.TheadvantageofSNPsinforensiccaseworkisthatsmallDNAfragmentsof40–50bpsfrome.g.heavilydegradedDNAcanbeSNPtyped.Furthermore,theSNPtechnologyhasahighpotentialforautomation.AlthoughthegeneticinformationobtainedbyaSNP,inaverage,ismuchlowerthanthatobtainedbyanSTRsystem,typingof50–100selectedSNPswouldbesufficientforforensiccasework[14].ThelowmutationrateofSNPs[15,16]makesthesemarkersanattractivetoolforparentagetesting.

GeneticmarkersontheYchromosomearevaluabletoolsinforensiccaseworkinspecialsituations,e.g.incaseswithmixturesofDNAwithadominantamountoffemaleDNAandaverysmallamountofmaleDNA.Insuchcases,theDNAprofileoftheautosomesofthemalecannotbeobtained,buttheYchromosomemarkerscanusuallybetyped,eveninsituationswithaverylargerelativeamountoffemaleDNA[17].Inspecialcasesofparentagetesting,e.g.

0379-0738/$–seefrontmatter#2003ElsevierIrelandLtd.Allrightsreserved.doi:10.1016/S0379-0738(03)00299-8

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–8475

iftheallegedfatherisunavailablefortestingwhileclosemalerelativesareavailable,investigationofgeneticmarkersontheYchromosomearevaluable.

IfSNPtypingisgoingtobeusedinforensiccasework,itisessentialthattheinvestigationscanbeperformedonsmallamountsofDNA,ifpossible,<1ngDNA.Ifthepolymerasechainreaction(PCR)isused,theamplificationsofallDNAfragmentstobeinvestigatedmustbedoneinoneorveryfewamplificationreactions.

WedecidedtoexploreaSNPtypingmethodthatisbasedonmultiplexPCRandmultiplexminisequencing.WechoseSNPmarkersontheYchromosomebecausethesemarkers,inforensicgenetics,offeradditionalinformationtotheinforma-tionobtainedbySTRtyping.Furthermore,theYchromosomeSNPsareusefultoolsforthestudyofgeneticsofpopulations.Inthelastyears,anumberofmultiplexPCRYchromo-someSNPanalyseshavebeenreported.MostofthemincludedalimitedamountofSNPs(often3–10SNPs)ineachPCR(e.g.[2,3,18])althoughlargermultiplexeshavebeenreported[19,20].

WeselectedYchromosomeSNPsthatwerereportedtobepolymorphicinEuropeanandotherpopulations[4,21,22].However,themainpurposeofthestudywastoexplorethetechnicalissuesrelatedtomultiplexingalargernumberofDNAfragmentsandsimultaneousdetectionofalargenumberofSNPs.TheintentionwasnottomakeafinalpanelfortypingofmajorYchromosomehaplogroups.InordertoassessthetechnicalperformanceoftheSNPtypingsystem,weincludedfourpairsofSNPseachofwhichpairwasexpectedtogiveconcordantresults(e.g.M40andM96).Here,wedescribeamethodfortyping35SNPsontheYchromosome.Thetypingwasperformedby(1)multiplexPCRamplificationof25YchromosomeDNAfragments,(2)multiplexprimerextensionreactionsof35SNPswithfluorescencelabellednucleotides,and(3)detectionofthe35SNPsbycapillaryelectrophoresisandmulticolourfluor-escencedetection.

2.Materialsandmethods2.1.DonorsandDNApreparations

Atotalof194unrelatedmalesand15unrelatedfemaleDanesdonatedbloodsamplesorbuccalcells.DNAwasisolatedfrom200mlofperipheralbloodusingQIAampDNABloodMiniKitaccordingtothemanufacturer’spro-tocol(Qiagen,Hagen,Germany).Alternatively,1.2mm(diameter)FTA1paper(WhatmanInternational,Cam-bridge,UK)soakedwithbloodorbuccalcellswasused.MixturesofDNAfrommalesandfemaleswerepreparedincheckerboardwiththreeconcentrationsofmaleDNA(0.16,0.8and1.6ng)andfemaleDNArangingfrom0to60ng.FluorometricmeasurementofDNAconcentrationwasdonebySYBRGreenIandanalysedinaLightCyclerinstrument(RocheDiagnosticsGmbH,Germany)and

Hoechst33258(MolecularProbesInc.,Eugene,OR)usingaHoeferDyNAQuant200instrument(MolecularVision).CalibrationreferencecurveswereestablishedusingacalfthymusDNAstandard(Sigma–Aldrich,Missouri,USA).2.2.SelectionofPCRamplificationprimers

TheYchromosomeSNPsselected(Table1)includedthoseusedbySeminoetal.[21]forastudyofthedistribu-tionofYchromosomeSNPsinEuropeanpopulations.Inaddition,weincludedSNPsthatwerereportedtobepoly-morphicinotherethnicgroups.

DNAsegmentsincludingtheSNPsselectedwereidenti-fiedandcomplementaryprimersweredesignedsothatthelengthsoftheamplifiedgenomicYchromosomeDNAfragmentswouldrangefrom79to186nucleotides.SomeSNPsweresituatedverycloselytoeachotheranditwasdecidedtoincludeanumberofamplificationtargetswithtwoorthreeSNPs(Table1).

ThesequenceofeachlocuswasobtainedfromGenBank1(http://www.ncbi.nlm.nih.gov)usinganucleotidebasiclocalalignmentsearchtool(BLAST).PublishedPCRprimerswereinitiallyusedasthereferencesequenceforeachYSNPlocus,butallofthemneededtoberedesigned.TheprimersforthegenomicsegmentsspanningoneormoreYchromosomemarkersweredesignedwiththePrimer3.0programv.0.2(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi).Allprimerswereselectedtohavetheoreticalmeltingtemperaturesof60Æ58Catasaltcon-centrationof180mMandapurine:pyrimidinecontentcloseto1:1,whenpossible.Thelengthsoftheprimersrangedbetween19and34nt.Primerswithfourormorebasesatthe30endcomplementarytoanotherpartoftheprimerwerediscardedorredesignedtoavoidartefactsduetohairpinformation.Eachprimerpairwastestedforprimer–primerinteractions,andtheprimersequenceswerecheckedtoavoidsimilaritieswithrepetitivesequencesorwithotherlociinthegenome.Theprimerswerecheckedforhomologytootherampliconsinthepoolof25primerpairs.Table1showsthesequencesoftheamplificationprimersselected.2.3.PCRconditions

HPLCpurifiedprimersforamplificationwerepurchasedfromTAGA/S(Copenhagen,Denmark).AprimerstocksolutionwaspreparedbydissolvingthelyophilizedprimersinTris/EDTAbuffer(10mMTris,100mMEDTA,pH7.5;Sigma–Aldrich)toafinalDNAconcentrationof100pmol/ml.EachprimerpairwastestedinsingleplexPCR.TenngtemplatewasamplifiedbyPCRina25mlreactionvolumecontaining1ÂPCRbuffer,1.5mMMgCl2,200mMofeachdNTP,0.4mMofeachprimer,and0.6unitsofAmpliTaqGoldDNApolymeraseat948Cfor5minfollowedby30cyclesof30sat958C,30sat608C,30sat728C,andafinalextensionfor5minat728C.Theproductswereanalysedbyelectrophoresisin11%polyacrylamidegels.

76J.J.Sanchezetal./ForensicScienceInternational137(2003)74–84

Table1

YchromosomeSNPsandprimersequencesforPCRamplificationof25YchromosomeDNAfragmentswithSNPsLocus

GenBankor

dbSNPsaccesionRs33Rs3900Rs3908Rs3909Rs3010AC009977AC009977Rs1179188AC006040Rs2032631AC002531AC009977AC0108Rs20320Rs2032652AC0108AC0108AC010137AC0108AC010137AC0108AC0108AC009977AC006040Rs2032597Rs2032604Rs2032624Rs2032678Rs20326Rs2032665AC0108Rs2534636AC005820Rs2535813Rs150173

Mutation

PCRprimers(50!30)Forwardprimer

A/GC/G4G/3G

2bpinsertionT/AT/CA/CG/CG/AG/AT/CA/CC/TC/TC/TG/CG/A5G/4GG/AT/AT/CA/CA/CC/TA/CT/GA/CÀ5bpC/AT/CT/CA/G

Present/absentGA/AC/CA

acggaaggagttctaaaattcaggaggaccctgaaatacagaactgcctggtcataacactggaaatccctggtcataacactggaaatccctggtcataacactggaaatctgaccgtcataggctgagacatgaccgtcataggctgagacaagggcatggtccctttctattggtctcaatctcttcaccctgt

gagagaggatatcaaaaattggcagttatatggactctgagtgtagacttgtgacctcaacttcccagagtgttgtgcattactccgtatgttcgac

catctcttaacaaaagaggtaaattttgtcctggattcagctctcttcctaaggttattgccctctcacagagcacttgttgcccaggaatttgcatccccgaaagttttattttattcca

catctcttaacaaaagaggtaaattttgtccccccgaaagttttattttattcca

catctcttaacaaaagaggtaaattttgtccgagagaggatatcaaaaattggcagtaggaccctgaaatacagaactgcggaaccactaccagcttca

cagctcttattaagttatgttttcatattctgtgtgagccctctccatcagaagttttcttacaattcaagggcatttaggatttaaactctctgaatcaggcacatccatataaaaacgcagcattctgttccatataaaaacgcagcattctgtttgcattactccgtatgttcgactcatccagtccttagcaaccattacactgactgatcaaaatgcttacagatttaaatccctcctatttgtgctaacctggaccatcacctgggtaaagt

Reverseprimer

aaaatacagctccccctttatcctaaatatttcaacatttcacaaaggaaagctgaccacaaactgatgtagaagctgaccacaaactgatgtagaagctgaccacaaactgatgtagattgaagcccccaagagagacttgaagcccccaagagagactccatgcagactttcggagtcatttcagtaaatgccacacaagatgacagtggcaccaaaggtcggtgccgtaaaagtgtgaaataatcgacgaagcaaacatttcaagagagtggaagcttaccatctttttatga

cattgtgttacatggcctataatattcagtctgctcaggtacacacagagtatcaccacccactttgttgctttg

cacagagcaagtgactctcaaagttctcagacaccaatggtcctatccattgtgttacatggcctataatattcagtttctcagacaccaatggtcctatccattgtgttacatggcctataatattcagttgacagtggcaccaaaggtcaaatatttcaacatttcacaaaggaaagttaaggccccacgcagt

gtcctcattttacagtgagacacaacgccaggtacagagaaagtttggctgaaaacaaaacactggcttatcattctactgatacctttgtttctgttcattctggagagaacttgagaaaaagtagagaatggagagaacttgagaaaaagtagagaatggaagcttaccatctttttatgaccacataggtgaaccttgaaaatgggatcccttccttacaccttatacaaatgcatgaacacaaaagacgtagaggcagtataaggttgtcacatcacat

0.150.360.090.090.090.070.070.420.180.030.460.030.080.240.030.270.020.060.240.060.240.030.360.030.070.160.100.020.120.120.080.060.060.040.01mM

Ampliconsize(bp)1281861701701701601609611913811515213217913514388113179113179138186113119179817917617613215090109

M2/sY81M9aM17aM18aM19aM32aM33aM35

M40/SRY40M45aM46/TatM52M78aM81aMM96M123M139aM151aM153aM1aM157aM163aM167/SRY2627M170M172M173M175M212aM213aM224aSRY10831/SRY153212f292R7P25

aSNPmarkersonthesameDNAfragment:(M9andM163),(M17,M18andM19),(M32andM33),(M45andM157),(M78andM224),(M81,M151andM1),(M139andM153),(M212andM213).Allprimerswereredesignedcomparedtopreviouslypublishedprimers.

TBE(1Â)(mmol/lTrisbase,mmol/lboricacid,2mmol/lEDTA,pH8.3)wasusedaselectrophoresisbuffer.Thegelswerestainedwith0.5mg/mlethidiumbromide.The10bpladderfrominvitrogen(Groningen,TheNetherlands)wasusedtoassignthesizesofthefragments.

ThefinalsetupofthePCRamplificationincluded1ngDNAina50mlreactionvolumecontaining1ÂPCRbuffer,8mMMgCl2,400mMofeachdNTP,0.01–0.42mMofeachprimer,and2.5unitsofAmpliTaqGoldDNApolymerase(AB,FosterCity,CA).

AllDNAamplificationswereperformedinaGeneAmp9600thermalcycler(Perkin-Elmer,Wellesley,USA)usingthefollowingprogramme:denaturationat948Cfor5minfollowedby33cyclesfor30sat958C,30sat608C,and30sat658C,followedbyafinalextensionfor7minat658C.Theconcentrationsoftheprimersinthemultiplexreac-tionwereadjustedinordertoobtainequalamountofeachPCRproduct.Theprimerconcentrationsrangedfrom0.01to0.42mM(Table1).

ThePCRproductswereanalysedon11%polyacrylamidegelsasdescribedlater(Fig.1).

InordertoeliminatetheexcessofprimersanddNTPs,thePCRproductswaspurifiedonaMinElutePCRpurificationspincolumn(Qiagen,Hagen,Germany)followingtheman-ufacturer’sprotocol.TheDNAwaselutedin30mlofMilli-Qwater.

E.coliexonucleaseI(ExoI)andshrimpalkalinephos-phatase(SAP)wasalsousedtoremoveprimersandunin-corporateddNTPs(USBCorporation,Cleveland,USA).SixmicrolitersExoSAP-ITkitor5unitsofSAPand2unitsof

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–8477

Fig.1.MultiplexPCRproductsof25YchromosomeDNAfragments.EthidiumbromidestainedpolyacrylamidegelwithPCRproductsobtainedfromvarioussourcesofblood.AnegativecontrolwithDNAfromafemalewasincluded.(L)10bpladderfrominvitrogene.

ExoIwereaddedto15mlofPCRproduct,mixed,andincubatedat378Cfor1h.Theenzymeswereinactivatedat758Cfor15min.

2.4.DesignofPCRminisequencingprimers

Table2showsthegenotypingprimersdesignedforeachSNP.Primersfordetectionofdeletionsandinsertionsweredesignedwiththe30,basecorrespondingtothelastbasebeforethepossibledeletionorinsertion.ForeachSNPsysteminvestigatedinthepresentstudy,thefollowingbasewouldidentifythepolymorphism.Thesequencesoftheprimerswerecheckedforthepossibilityofprimer–dimerandhairpinformationandinvestigatedinPCRwithouttemplate(‘self-extensionreaction’).Inordertodistinguishbetweenthesizesofthedetectionprimers,theprimersweresynthesizedwithlengthsbetween19and106nucleo-tideswithintervalsoffournucleotidesforthegreatmajor-ityoftheprimers(Table2).Thelengthsofthetemplatespecificpartsoftheprimersrangedfrom16to29nucleo-tides.Thedesiredlengthofaprimerwasadjustedatthe50endbyadditionofapieceofa‘neutral’sequenceand,ifnecessary,apoly-Ctail.Theneutralsequence,

50-AACTGACTAAACTAGGTGCCACGTCGTGAAAGT-CTGACAA-30,isarandomsequencethatdidnotmatchwithanyhumansequenceintheNCBInon-redundantdatabase[19].

Foreach4bpDNAfragmentsizeintervalofthedetectionprimers,twoSNPlociweredetected.ThiswasdonebyselectingtwoSNPlociwithdifferentnucleotidepolymorph-ism.OneSNPcouldbe,e.g.anA/TSNPandtheotheraC/GSNP.Thus,theminisequencingprimersforthetwoSNPscouldhavethesamelengthandthetwopolymorphismswouldstillbedetectable.PrimersforminisequencingwereHPLCpurified(DNA-TechnologyA/S,Aarhus,DenmarkandProligoFranceSAS,Paris,France).2.5.Minisequencingreactionandcapillaryelectrophoresis

MultiplexPCRminisequencingwasperformedin8mlreactionswith0.2mlpurifiedPCRproduct(6–10ngequiva-lentto5–8fmolofeachfragment),4mlofSNaPshotTMreactionmixand0.01–0.5mMoftheprimers(Table2).Thethermalcyclingwasperformedwitharapidthermalrampto968Cfor10s,508Cfor5s,and608Cfor30sfor25cycles.

78J.J.Sanchezetal./ForensicScienceInternational137(2003)74–84

Table2

Minisequencingprimersequencesfortypingof35YchromosomeSNPmarkersLocus

M170M45M139M2/sY81M46/Tat

M167/SRY2627M213M52P25M72R7MM123M35M153

M40/SRY40M1M32M151M17M96M172M173M19M224

SRY10831/SRY1532M18M157M81M163M212M912f2M33M175

aPoly(dC)NoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNoneNone734710171814252022292946

NeutralSequence(50!30)

NonecaaaagacaaNone

tgaaagtctgacaatgacaatctgacaa

tcgtgaaagtctgacaagaaagtctgacaagtgaaagtctgacaa

cacgtcgtgaaagtctgacaaacgtcgtgaaagtctgacaa

ggtgccacgtcgtgaaagtctgacaaggtgccacgtcgtgaaagtctgacaa

aaactaggtgccacgtcgtgaaagtctgacaagccacgtcgtgaaagtctgacaataggtgccacgtcgtgaaagtctgacaaaggtgccacgtcgtgaaagtctgacaa

actaaactaggtgccacgtcgtgaaagtctgacaa

aactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaaaactgactaaactaggtgccacgtcgtgaaagtctgacaa

Targetspecificsequence(50!30)

caacccacactgaaaaaaactcagaaggagctttttgctaatctgacttggaaaggggctttatcctccacagatctca

gctctgaaatattaaattaaaacaacaagccccacagggtgc

tcagaacttaaaacatctcgttacaatatcaagaaacctatcaaacatcctgcctgaaacctgcctg

cttattttgaaatatttggaagggccatgaacacaaaagacgtagaagaactcaggcaaagtgagagatatttctaggtattcaggcgatgtcggagtctctgcctgtgtcgctcaaagggtatgtgaacatccaccctgtgatccgct

gttacatggcctataatattcagtacaagacaagatctgttcagtttatctcacaatctactacatacctacgctatatgccaaaattcacttaaaaaaacccggaaaacaggtctctcataatacaaacccattttgatgctt

tacaattcaagggcatttagaacaaactatttttgtgaagactgttgtaaattgatacacttaacaaagatacttcttgtatctgactttttcacacagtgtttgtggttgctggttgttacaccaaaggtcatttgtggt

cttggtttgtgtgagtatactctatgaccacaaaggaattttttttgag

gcattctgttaatataaaacacaaaacatgtctaaattaaagaaaaataaagagaacatgtaagtctttaatccatctccagttacaaaagtataatatgtctgagatcacatgccttctcacttctc

OrientationamMReverseReverseForwardReverseReverseForwardReverseReverseForwardReverseReverseReverseReverseReverseForwardReverseReverseForwardForwardReverseForwardForwardForwardForwardForwardForwardForwardReverseReverseReverseForwardReverseForwardReverseForward

0.020.020.010.280.250.350.020.020.040.020.010.090.030.250.020.080.030.500.020.020.150.100.030.100.130.030.050.200.030.510.200.400.020.180.28

Primersize(nt)19222226263030343438384242465050586266667074747878828686909498106

ThedetectionorientationhasbeenprobedrelativetotheYCCinformationreportedin[32].

Apositivecontrol(providedwiththekit)andnegativecontrol(sterilewaterorPCRproductfromafemale),wasperformedforeachbatchof44samples.

Thehomogeneityofeachprimerwascheckedinsingle-plexminisequencing.Theoccurrenceofextrapeaksoneormorenucleotidessmallerthantheexpectedsizeindicatedheterogeneityoftheminisequencingprimer.

Aftertheminisequencingreaction,1UnitofSAPwasaddedandthetubewasincubatedat378Cfor1hinordertoremovethe50phosphorylgroupsoftheunincorporated[F]ddNTPs.SAPwasinactivatedbyincubationat758Cfor15min.

OnemlofthepurifiedminisequencingPCRproductwasanalysedonanABPrism3100GeneticAnalyserwitha36cmcapillaryarray,POP-4polymerand10sat3000V

injections.GeneScan-120LIZTMwasusedasinternalsizestandard.ThedatawereanalysedusingGeneScanAnalysissoftwarev.3.7(AppliedBiosystems).Afterbackgroundsubstractionandcolourseparation,peaksweresortedintobinsaccordingtosizesbycomparisontotheinternalsizestandard.Peaksabove400relativefluorescenceunitswereconsideredpositivesignalsandaSNPtypewasassigned.

2.6.Reproducibilitystudies

DNAsamplesfrom194unrelatedmaleDanesweretypedtwicewiththeminisequencingtechniqueandassignedSNPtypesforthe35SNPsystems.TheassignmentsofSNPtypesoftheduplicatetestingwerecompared.

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–8479

2.7.Statisticalmethods

GenediversitiesandstandarderrorswerecalculatedaccordingtothemethodsofNei[23].3.Results

3.1.DNApurificationmethods

DNApurifiedwithQiagencolumnsandDNAfromFTA1paperwithbloodstainsinallcasesgavesatisfactoryresults(Fig.1).DNAfrombuccalcellsonFTA1papergavevariableintensitiesoftheresultsofsamples.3.2.Designofprimers

Whennobandoronlyaveryweakbandwasobserved,suggestingthattheaffinitiesoftheprimersweresuboptimal,theprimerswereredesigned.Inonecase,thePCRamplifica-tionwasveryweakandfourdifferentsetsofprimersweretriedbeforeanacceptableyieldwasobtained.Itwasnotpossibletounderstandthereasonsincetheprimersetbestsuitedfromatheoreticalpointgavethelowestyield.Inthreecaseswithunsatisfactoryyields,theprimerswereredesigned

with‘GC’atthe30endwithsuccessfulresults.Twenty-oneofthe25primerpairsworkedsatisfactorilyatthefirstdesign.3.3.PCRbufferandefficiencyofmultiplexPCRamplification

Wefoundthatthebestresultsofamplificationofall25DNAtargetswereobtainedbyincreasingtheconcentrationto8mMMgCl2.Higherconcentrationsinhibitedtheampli-fication(datanotshown).

3.4.QualityofDNAprimersfortemplatePCRamplification

UnpurifiedprimerscouldbecombinedintomultiplexesuptosevensystemswhileHPLCpurifiedprimerscouldbecombinedtoamplifyatleast25templatesinonereaction.3.5.TitrationofprimerconcentrationsinPCRamplification

ItwasnecessarytotitrateprimerconcentrationstoobtainabalancedPCRmultimixforminisequencing.Thefinalconcentrationsofprimersrangedfrom0.11to0.46mM.

Fig.2.Electropherogrammewith35YchromosomeSNPprofilesfromamaledonor.GeneScananalysisofSNaPshotTMminisequencingoftheYchromosomeSNPmultiplex.

80

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–84

25)%(stin20U ecne15cseroul10F evita5leR050

100

250

5001000200040008000

DNA (pg)

Fig.3.Sensitivityofthe35YchromosomeSNPtypingassay.ForeachDNAconcentration,therelativefluorescenceunits(RFUsfromGeneScan)ofinvestigationsoffourSNPsdetectedwitheachofthefourdyes:blue,green,yellowandredwerecollatedfromtypingoftwoindividuals.ForeachDNAconcentration,themedianRFUvalueofthetwoindividualswascalculatedforeachdye,andforeachconcentrationthemedianRFUswerenormalizedasapercentageofthetotalRFUsofalltheRFUsforthedyeinquestion.Finally,foreachDNAconcentration,themedianofthenormalizedRFUvaluesforallfourdyeswascalculatedasapercentageofthesumofallnormalizedmedianRFUvaluesofallconcentrations.Thus,thesumofRFUsinthefiguresumupto100%.Theerrorbarsindicatethestandarderrorofthemean(S.E.M.).

3.6.SensitivityofthetargetmultiplexPCRamplificationInourhands,thelowerlimitforreproducibleresultswasapproximately100pgDNAwitharangeuptoapproxi-mately10ngandanoptimumat1–2ng(Figs.2and3).3.7.PurificationofthePCRtemplateamplificationproduct

BothspincolumnandenzymaticpurifiedPCRamplifica-tionproductsgavesatisfactoryminisequencingtypingreac-tions.TherecoverywiththeExoI-SAPwas100%whilethecolumnpurificationhadarecoveryofapproximately80%(datanotshown).

3.8.DesignofDNAprimersforminisequencing

Noneofthe35detectionprimershadtoberedesigned.3.9.QualityofDNAprimersforminisequencing

Clear,homogeneouspeakswereobtainedonlyifthepurityoftheprimerswashigherthanapproximately90%.Ifthepuritywasless,thesignalfromdegeneratedprimers(nÀ1,nÀ2,etc.)woulddecreasethediscrimination.

3.10.Annealingtemperatureofminisequencingprimers

Annealingtemperaturesfrom50to608Cgavealmostthesameoverallresultsinthe35SNPmultiplexwhenjudgedbyinspectionofthepeakareas.

3.11.YchromosomeSNPtypingresults

Fig.2showsarepresentativeelectropherogrammeoftypingof35YchromosomeSNPsinanindividual.Inoneofthe194males,typingcouldnoreactionwasobtainedinM81.ThesamelackofreactioninM81wasfoundinthesonoftheinvestigatedman.Theremaining34Ychromo-someSNPsweredetectedinthemanandhischild.Allothermalesamplesgaveafull35-Y-SNPprofile.3.12.ReproducibilityofYchromosomeSNPtypingwithminisequencing

SNPtypingwasperformedtwiceinall194maleDanesandtheduplicatetypeswereconsistent.Ineachminisequen-cingexperiments,atleastonesamplewithknowntypesforall35SNPswasincluded,andconcordantassignmentsofSNPtypeswereobtainedinallcases.

Foursamplesweretypedforthe11SNPsSRY2627,M213,M35,M153,SRY40,M17,M18,M9,SRY10831,92R7,andP25aspartofaninterlaboratoryexerciseoftheEuropeanDNAProfilingGroup,andcorrectresultswereobtained.

Fig.4.EffectofexcessDNAfromfemalesonthe35YchromosomeSNPtypingassay.Therelativefluorescenceunits(RFUsfromGeneScan)ofmixturesofmaleDNAandfemaleDNAingreatexcess.TheRFUswerecalculatedasindicatedinFig.3.Ingeneral,therewasadoseresponserelationbetweentheconcentrationofmaleDNAandtheRFUsignalstrength,whilefemaleDNAhadpracticallynoinfluenceontheRFUsignalintheconcentrationrangeinvestigated.

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–84

Table3

Frequenciesof35YchromosomeSNPmarkersinmaleDanesLocus

M2/sY81M9M17M18M19M32M33M35

M40/SRY40M45M46/TatM52M78M81cMM96M123M139M151M153M1M157M163

M167/SRY2627M170M172M173M175M212M213M224

SRY10831/SRY153212f292R7P25

ab81

Fragmentnumbera123334456710111213141511151172161718192021211022232425

PolymorphismbA/GC/G4G/3G

Noins./2bpins.T/AT/CA/CG/CG/AG/AT/CA/CC/TC/TC/TG/CG/A5G/4GG/AT/AT/CA/CA/CC/TA/CT/GA/C

Nodel./5bpdel.C/AT/CT/CA/G

Present/absentGA/AdC/CAdFrequency(number)194/085/109162/32194/0194/0194/0194/0190/4190/486/108193/1194/0192/2193/04/190190/4193/10/194194/0194/0194/0194/0194/0194/0119/751/5/105194/0194/04/190194/032/1621/586/108124/70

Frequency(%)100.0/0.043.8/56.283.5/16.5100.0/0.0100.0/0.0100.0/0.0100.0/0.097.9/2.197.9/2.144.3/55.799.5/0.5100.0/0.099.0/1.0100.0/0.02.1/97.997.9/2.199.5/0.50.0/100.0100.0/0.0100.0/0.0100.0/0.0100.0/0.0100.0/0.0100.0/0.061.3/38.797.4/2.5.9/.1100.0/0.0100.0/0.02.1/97.9100.0/0.016.5/83.597.4/2.4.3/55.763.9/36.1

SomePCRproductscontainmorethanoneSNPinthesamefragment.FollowingtheYchromosomeconsortiumnomenclaturesystem[32].cOnemalegavenoreactioninminisequencingofM81.dTwosignalsweredetectedinsomeindividuals[24,33].

3.13.Male–femalemixturesofDNA

FemaleDNAdidnotinfluencetheresultsofYchromo-someSNPtypingwhenaddedinconcentrationsmorethan300timestheconcentrationsofmaleDNA(Fig.4).3.14.YchromosomeSNPpopulationdatainDanesTable3showsthefrequencydistributionofthe35SNPsinvestigatedin194maleDanes.NoSNPsignalwasobtainedin15femaleDanes.Atotalof19SNPsshowedvariationwhile16SNPsweremonomorphicinthemaleDanesstudied.

TwosignalswereobtainedinP25and92R7insomeindividuals(cfdiscussion).DNAfromindividualswithtwo

signalsinthesessystemswasinvestigatedwithSTR-tech-nique.OnlyoneSTR-profilewasobtainedineachindividualdemonstratingthatcontaminationofDNAwasnotthereasonforthetwosignalsinP25and92R7.

4.Discussion

WehavedevelopedaPCRmultiplex-basedsystemfortypingofalargenumberofSNPsusingYchromosomeSNPsasanexample.AnimportantpartoftheworkwastoexplorethevariousaspectsofthemultiplexPCRmethods.The35YchromosomeSNPspresentedherearenotourfinalsetofYchromosomeSNPsforpopulationstudiesorforensicgeneticapplications.

82J.J.Sanchezetal./ForensicScienceInternational137(2003)74–84

SuccessfulPCRmultiplexingdependsonanumberoffactors.Below,wepresentsomeofourconsiderationsconcerningtheselectionoftheSNPsandthegenerationofthemultiplexPCRsforamplificationandminisequen-cing.

Atanearlystage,itwasdecidedtousethemulticolourfluorescenceelectrophoresistechniquecombinedwithPCRmultiplexingatapproximately608CinhighconcentrationsofMgCl2.Thespacingbetweenminisequencingprimerswasdecidedtobefournucleotidesbecausewewantedtoobtainreliableseparationintheelectrophoresis.

WeattemptedtoavoidSNPssituatedinregionsreportedtobereplicated.TwoexceptionsweretheP25and92R7SNPsthataresituatedinaregionthatmostprobablyispartofaduplication[24].BothSNPsseemtodiscriminatebetweenEuropeanandotherpopulations[25].

MultiplexPCRamplificationprimersbetween19and34basespairslongwereselectedbecauseitwasanticipatedthatsuchlongprimerswouldworkwellundermultiplexcondi-tions[26].

QiagenpurifiedDNAfrombloodsamplesandbloodstainsonFTA1paperworkedequallywellintheassay.Chelextreatedbloodsamplesworkedaswell(datanotshown).OptimalmultiplexSNPtypingresultswereobtainedwith1ngDNA(range0.1À20ngDNA).Thus,quantifica-tionofDNAisnotmandatoryfortheSNPassay.Itshould,however,benoticedthatthebalanceoftheamountsofamplificationproductsoftheDNAfragmentsischangedwithincreasingamountsoftemplates.WithincreasingconcentrationsofPCRamplifiedfragments,small,fluores-centadenosinnucleotidepeakswithsizesofPCRamplifiedfragmentsplusonenucleotidewereseen,mostlikelydotonon-templateadditionofasingleadenosinmoleculestothe30endofsomePCRamplifiedfragments.AtlowamountsoftemplateDNA,lossofsignalwilloccurduetostochasticphenomena[27].

CommonlyusedPCRbuffersincludeonlyKCl,TrisandMgCl2.Ithasbeenreportedthatmanyprimerpairsprodu-cingshortamplificationproducts(<200bp)workbetterathighersaltconcentration(KCl)inmultiplexsystems[26].IncreasingtheconcentrationofKClinthePCRbuffer1.6and2-foldinour35-plexdidnotincreasetheyieldofPCRproductsignificantlyandhadnoeffectonthesynthesisoffragments>150bp.IncreaseofMgCl2concentrationfrom2to8mMincreasedtheyieldofamplicons;higherMgCl2concentrationinhibitedtheamplification(datanotshown).WeusedAmpliTaqGoldDNApolymerase(AppliedBiosystems)becausethisenzymeminimizesprimerdimerformation.Evenwitha4-base30overlapbetweentwoprimersweobtainedhomogeneousPCRproducts(datanotshown).Themostefficientenzymeconcentrationseemedtobearound2.5U/50mlreactionvolume.

Inourhands,primerconcentrationsbelow0.01mMwereinsufficientandconcentrationsabove0.5mMseemedtoinhibitmultiplexPCRSprobablybyinducingdimer–dimerformation.Primerconcentrationswereadjustedtobe

approximately103timesmorethantheconcentrationofthetemplate.

WestoreddNTPsinsmallaliquotsatÀ208Cforupto8months.However,weobservedthatdNTPsweresensitivetorepeatedfreezingandthawing.Asaruleofthumb,themultiplexPCRwouldfailifthedNTPshavebeenfrozenandthawedmorethanfourtimes.Theamountoftimeinfreezerwaslessimportantasithasbeenreportedbyothersauthors[28].

Theenzymaticpurificationmethodisobviouslyeasy,hasanalmost100%recoveryandaverylimitedriskofcon-tamination.

Wechosetoadjustthelengthoftheminisequencingprimersbymeansof(1)apartofaneutralsequenceofupto40ntandforthelongerprimers(2)anadditionalpoly-Cpart.Theneutralsequencewasselectedinordertoobtainamorebalancedbasecomposition.Wechosepoly-Cforthetailbecause,intheory,poly-Gwouldgiveahighermolecularmass,poly-Awouldhaveariskofdepurinationduringsynthesis,andpoly-Ttailsmayinterferewiththeadditionof30ddAintheminisequencingreaction(SNaPshotTMprotocolrecommendation,AppliedBiosystems).

ThequalityofminisequencingprimersisimportantbecauseprimerbatcheswithheterogeneousprimersequencesconsistingoftheintendedDNAsequenceof‘n’nucleotidesplusaspectrumofshorternucleotides(nÀ1,nÀ2,nÀ3,etc.)inmanycaseswilldestroytheminisequencingreaction.Inaddition,weobservedamplifi-cationfailureduetoaheterogeneousprimerbatchinthePCRmultiplexwithsevensystemseventhougheachofthesevenworksinsingleplexreactions.Therefore,werecom-mendthateachprimerbatchistestedbeforethemultiplexPCRandsubsequentanalyses,e.g.byminisequencingormassspectrometry.Purificationoftheprimerswithe.g.HPLCorgelpurificationtechniquescantosomeextentsolvetheseproblems.

Theminisequencingsystemwasratherinsensitivetotheannealingtemperature.Itwasnecessarytoadjustprimerconcentrationsfrom0.01to0.50mMintheminisequencingmultimix.

Thelongerextensionproductshadelectrophoreticmobi-litiescorrespondingtothosepredictedbythenumberofbases.Themobilityofshorterextensionproductswiththesamenumberofbasesvariedtosomeextent.ThisismostprobablyduetothefactthatdifferencesinthemassesofthevariousfluorochromesusedandintheexactcompositionofpurinesandpyrimidineshavearelativelyhighinfluenceonthemobilityofshortDNAmolecules.

TheSNP-typingresultswerehighlyreproducible.Atotalof194maleswereSNPtypedinduplicateandnodiscre-pancieswereobserved.Furthermore,fiveofthemostpoly-morphicSNPswereanalysedbyaDNAhybridisationassayusingtheNanogentechnology[29].Concordantresultswereobtainedforall194individuals(datanotshown).

Inonefather-childcombination,noalleleofM81wasdetectable.Anamplifiedfragmentwaspresentinthefirst

J.J.Sanchezetal./ForensicScienceInternational137(2003)74–8483

PCRbecausetwootherSNPs(M151andM1)onthefragmentweredetected,butnoreactionofM81wasdetectedintheminisequencingreaction.Workisinprogressinordertodeterminethenatureofthevariant.

Atotalof19of29SNPsreportedtobepolymorphicinEuropeansinapreviousstudy[4]and9of10SNPsreportedinanotherstudy[21]turnedouttobepolymorphicinthemaleDanesstudied.Thegenediversityforthelocishowingpolymorphismrangedfrom0.01to0.5(Table3).M173,M45,92R7andM9werethemostpolymorphicmarkersinDanes.ThedataweredescribedasfrequenciesofindividualSNPsandnotasYchromosomehaplogroupsbecausethestudywasatechnicalstudyandtheYchromosomemulti-plexisnotidealfortypingofYchromosomehaplogroups.AlargerstudyofYchromosomehaplogroupsinDanesandotherpopulationswillbepublishedelsewhere.

P25and92R7werepreviouslyreportedasSNPs[30,31].However,theP25and92R7minisequencingprimerswereextendedwithtwodifferentdideoxynucleotidesduringtheminisequencingreactionofnumeroussamples.Thisindi-catesthatatleasttwodifferent,almostidenticalfragmentswereamplifiedduringthePCRreaction.Hurlesetal.[33]previouslyobservedthatSNPtypingof92R7gavetworesultsinsomeindividuals.FurtherstudieshaveconfirmedthatP25and92R7areparalogoussequencevariantsandthatatleastoneofthesequencevariantsineachgroupoflociispolymorphic[24].

ThemultiplexPCRSNPtypingformatpresentedhereseemstobeusefulforforensiccaseworkbecausesmallamountsofDNA(100pgDNA)canbereliablytyped.ThemultiplexpresentedisnotourfinalpackageforYchromosomeSNPsforforensicpurposes.Thewayforwardwouldgoeitherthrough(1)thedevelopmentofSNPpackagesoptimisedforaninitialscreeningplusfurtherpackagesoptimisedforthemajorpopulationsor(2)thedevelopmentofalargemultiplexpackagethatincludeYchromosomeSNPsthatcandiscriminatebetweenindividuallineagesinallpopulations.

Acknowledgements

WethankDr.RebeccaReynolds,RocheMolecularSys-tems,foradviceconcerningthedesignofthemultiplexPCRfortemplategenerationintheinitialphaseoftheproject.WethankMs.AnneMetteHolboBirkfortechnicalassistance.TheworkwassupportedbygrantstoJuanSanchezfromEllenandAageAndersen’sFoundationandManuelMoralesFoundation.References

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