生物质谱作为SNP分型检测方法的研究

:2003202217;:2003209209:863(2001AA224011):(1974),(),,,E-mail:[e-mail]3:,,:010266932025,E2mail:[e-mail]244200310JournalofChineseMassSpectrometrySocietyVol.24No.4Oct.2003SNP1,2,1,3,1,2,1,1,3(1.,100850;2.,100176;3.,100850):MillerDNA(Polymerasechainreac2tion,PCR),(SNP)DNA,CIPEoxIPCR(dNTP),SNP2(MALDI2TOFMS)2(ESI2QqTOFMS),:;(SNP);2(MALDI2TOFMS);2(ESI2QqTOFMS):O657.63;Q524.3:A:100422997(2003)042449207,DNA,DNA,(Singlenucleotidepolymor2phism,SNP)DNA,1%,DNA90%SNP[1]SNP,,SNP:,SNP(SNPmapping);,SNP,SNP(SNPgenotyping);,,SNP(),,,SNP,2080,(ElectrosprayIonization2MassSpectrometry,ESI2MS)(Ma2trix2AssistedLaserDesorptionIonization2MassSpectrometry,MALDI2MS),[2],SNP,MALDI2TOFMSMALDI2TOFMSSNP,ESI2QqTOFMSSNP,SNP1111ReflexËMALDI2TOFMS:Bruker;Q2TOFmicro2:Micromass1.2MillerDNA;;Hot2StarTaqDNA:QIAGEN;(dNTPs)(ddNTPs)(Themosequenase):Amersham;I(Exonucle2asesI,ExoI)(Calfin2testinalalkalinephosphatase,CIP):Bio2labs;32(32hydroxypicolinicacid,HPA)(Trihydroxyace2tophenone,THAP),GenopureKits:Bruk2er1.31.3.1PCRPrimer3,1(PCR)(10LL):5ngDNA,0.2mol(),0.05(U)HotStarTaqDNA,3.0mmolMgCl2,300Lmol(dNTPs),1buffer:9515min(DNA),9430s,55.530s,7230s,35PCR2.0%1.3.2(dNTPs)PCR1.5(U)(CIP)1(U)I(ExoI),3745min,8515min,CIPExoI1.3.325pmol,20.5(U)(Thermosequenase),100Lmol(ddNTPs),2LLThermosequenasebuffer(10),20LL9420s,5040s,6020s,351.3.4Genopurekit,1.3.5MALDI2TOFMS:0.5LL0.5LL(2,4,62THAP200mol(50%),2,3,42THAP100mol(50%),50mol),,0.5LL384,,200ns,19kV(ISö1),16.45kV(ISö2)(200fmol,Mr=5733.6Da)1.3.6ESI2QqTOFMS2LL,2LL0.5LL250mmol(V()V()=41)250mmol(V()V()=11),,NaI(5mgöL)Cone30V,möz4001500,80054241PCRTable1PrimersusedinPCRsystem(Gene)(Forwardprimersequence)(Reverseprimersequence)PCR(PCRproductlength)GAPDgccaaaagggtcatcatctcttcacacccatgacgaacat62IL12RB1aatatcagcgtcggaaccaactgccattcaatgcaatacg81TNFR1ctgtccctcgtcacttcctcttctgtttctcctggcctgt97LTAaatgaggtgagcagcaggttagccaaaccttgagcccta153TNFRSF9ggctgcaagtggaattagacattgacttgccgtgtgact174IL10RBtaccacctcccgaaaatgtccagactcccttcctcctgtg202TNFRSF5gggaatgttctggggaaactggggcaaaaacaactcacag2532Table2Extensionprimersequencesandmassesforunextendedandextendedproducts(Gene)(Extensionprimerssequence)öDa3(Massofunextendedprimers)(Variantalleles)öDa(Massofextendedprimers)GAPDatctctgccccctctgctga5979.90TöC6268.10ö6253.10IL12RB1gccattcaatgcaatacgtc6061.01AöG6358.23ö6374.23TNFR1gcggcacagctaaaggag5582.69AöG5879.90ö5895.90LTAagcagcaggtttgagg4986.31GöT5299.52ö5274.51TNFRSF9ggttccaaccctgc4199.77TöC4487.97ö4472.97IL10RBgtgggagtcacctgcttttgcc6733.41AöG7030.62ö7046.62TNFRSF5cctggtctcacctcgc4769.12TöC5057.32ö5042.32(Note):3:v1.1,ddNTP(ddA:297.2148;ddG:313.2097;ddC:273.1898;ddT:288.2015)(3:Molecularweights(Mr)ofprimersaremeasuredbyanon2linesoftwarenamedMolecularMassCalculatorforoligonucleotidesv1.1(http:ööthor.prohosting.comöjef_röcgi2binömololi.cgi).ExtendedprimersaremeasuredbyaddingddNTP’sMrtotheMrofunextendedprimer(ddA:297.2148;ddG:313.2097;ddC:273.1898;ddT:288.2015))22.1MALDI-TOFMSESI-QqTOFMSMALDI2TOFMSESI2QqTOFMS,141MALDI2TOFMS,2ESI2QqTOFMS,a,b;3ESI2QqTOFMS4MALDI2TOFMS(a)ESI2QqTOFMS(b)7SNPSNP,[3],MALDI2TOFMS,ESI2QqTOFMS,,,,MALDI2TOFMS,1544,SNP1SNPMALDI-TOFMS(3)a;bFig.1MassspectraofMALDI-TOFMSforSNPgenotyping(Peakslabeledwith3meanproductcausedbybaselosing)aHomogeneous;bHeterozygous2SNPESI-QqTOFMSa;bFig.2MassspectraofESI-QqTOFMSforSNPgenotyping(Transformedbythesoftware-MaxEnt1)aMulti2chargedspectrum;bSingle2chargedspectrum3SNPESI-QqTOFMS(3)a;bFig.3MassspectraofMALDI-TOFMSforSNPgenotyping(Peakslabeledwith3meanadductsformedwithalkalications)aOligonucleotidesinammoniumsolutionsystem;bOligonucleotidesinpiperidineandimidazolesolutionsystem254244SNPMALDI-TOFMSESI-QqTOFMS(3)aMALDI2TOFMS;bESI2QqTOFMSFig.4MALDI-TOFMSandESI-QqTOFMSspectraofseven-plexSNPgenotyping(Peakslabeledwith3meanproductcausedbybaselosing)aSpectrumofMALDI2TOFMS;bSpectrumofESI2QqTOFMS(Peakslabeledwith3meanpeaksformedbybaselosing)2.2MALDI-TOFMSESI-QqTOFMSSNPSNP:SNP,,,;,,[4]SNP,,,()[5],,;,SNP,4,SNP,SNP[6,7][8][9][10],[5]MALDI2TOFMS,1min,,,,,(Sweethot),,,MALDI2TOFMS,,,,(A)(T)(,9Da),45,SNPSNP12[11]MALDI2TOFMS,ESI2MSMALDI2TOFMSSNP[1214],ZhangS[12]2,ddNTPsWaltersJ[13]2PCRSNPDNAKrahmerM[14]2PCR,62DNA2,MALDI2TOFMS,,3544,SNPESI2QqTOFMS,,SNPESI2QqTOFMSSNP87SNP,ESI2QqTOFMSMALDI2TOFMS,MALDI2TOFMS,ESI2QqTOFMSSNP(),,ESI2QqTOFMS,(HPLC),,MALDI2TOFMSKrahmerM[14],ESI2QqTOFMSSNPESI2QqTOFMS,SNP,,ESI2QqTOFMSSNP,ESI,,,TNFR1McLuckeyS[15](IonöIon),ESI,,ESI2QqTOFMS,MALDI2TOFMS,[15],,,3SNPSNPSNPDNA,CIPEoxIPCRdNTP,SNP(MAL2DI2TOFMSESI2QqTOFMS)SNPESI2QqTOFMS,MALDI2TOFMS,MALDI2TOFMSESI,MALDI2TOFMS,,,SNPESI2QqTOFMS,SNP,,,,,,,(),:[1]BrookesAJ.TheEssenceofSNPs[J].GENE,1999,234:177186.[2].21[M].:,1999:112113.[3]YangH,WangH,WangJ,etal.MultiplexSin2gleNucleotidePolymorphismGenotypingbyMa2trix2assistedLaserDesorptionIonization2Time2of2FlightMassSpectrometry[J].Analbiochem(InPress).[4]GutIG.AutomationinGenotypingofSingleNe2cleotidePolymorphisms[J].HumanMutation,2001,17:475492.[5]LechnerD,LathropGM,GutIG.Large2scaleGenotypingbyMassSpectrometry:Experience,AdvancesandObstacles[J].CurrOpinChemBio,2001,6:3138.[6]Grif