三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用

HEREDITAS(Beijing)20105,32(5):517―523ISSN0253-9772技术与方法收稿日期:2009−10−27;修回日期:2010−01−09基金项目:(:[2008]86)作者简介:(1982−),,,:Tel:[phone];E-mail:[e-mail](1983−),,,:Tel:[phone];E-mail:[e-mail]通迅简介:(1964−),,,,:Tel:[phone];E-mail:[e-mail]:10.3724/SP.J.1005.2010.00517三酶焦测序体系的建立及其在唐氏综合征快速诊断中的应用刘夕群1,朱术会2,邹秉杰2,马寅姣2,周国华1,21.,210029;2.,210002为了避免四酶焦测序体系中由于三磷酸腺苷双磷酸酶(apyrase)造成的测序结果偏差,文章建立了一种定量性能好的无三磷酸腺苷双磷酸酶的三酶焦测序体系。方法是将生物素修饰的DNA模板、荧光素酶和ATP硫酸化酶固定在磁性微球表面进行焦测序反应,当加入一种dNTP进行焦测序反应完后,采用磁性分离技术,除去焦测序反应产生的ATP和剩余的dNTP,然后加入另一种dNTP进行测序,按同样的方法去除影响下一轮测序反应的成分,实现循环测序。此体系能准确判读待测DNA的碱基序列,且可定量测定单核苷酸序列多态性(SNP)中两种等位基因型的相对比值。文章成功检测了16例正常人和8例唐氏综合征患者样本中21号染色体上两个杂合率较高位点(rs1042917和rs4818219)的等位基因型比值,所得结果能够明确说明待测样本中来自于父方和母方的21号染色体数目是否相等。该法具有良好的定量性能,适合于SNP等位基因型的定量分析,可以用于唐氏综合征的快速检测。腺苷双磷酸酶;三酶焦测序体系;唐氏综合征;单核苷酸序列多态性Developmentof3-enzymepyrosequencingsystemanditsapplicationinrapiddiagnosisofDown’ssyndromeLIUXi-Qun1,ZHUShu-Hui2,ZOUBing-Jie2,MA-Yin-Jiao2,ZHOUGuo-Hua1,21.SchoolofBasicMedicalSciences,NanjingMedicalUniversity,Nanjing210029,China;2.HuadongInstituteforMedicineandBiotechnics,Nanjing210002,ChinaAbstract:Toavoidsequencingerrorresultingfromuseofapyraseinconventional4-enzymepyrosequencingsystem,anon-apyrase3-enzymepyrosequencingsystemwithabetterperformanceofquantitativeanalysiswasestablished.ThemethodistoimmobilizebiotinylatedDNAtemplate,ATPsulfurylaseandluciferaseonstreptavidin-coatedmagneticbeadsforpyrosequencing.Afterpyrosequencing,ATPproducedfromthepyrosequencingreactionandexcessdNTPswerere-movedbymagneticseparationtechnique;anotherdNTPwasthendispensedforsequencingreaction,andthecomponentsinterferingwiththenextcircleofpyrosequencingreactionwereremovedbythesameway,achievingthecircularsequenc-ing.ThisnewsystemcanaccuratelymeasurebasesequencesofatargetDNAtemplate,andalsocanquantitativelydeter-minetherelativeratiooftwoalleles.ThealleleratiosintwoSNPs(rs1042917andrs4818219)havingahigherheterozygote518HEREDITAS(Beijing)201032rateonchromosome21weresuccessfullydetectedfor16normalsamplesand8clinicalsamplesfromDown’ssyndromepatients.Theresultscanaccuratelydemonstratewhetherornotthetargetsamplehasequalcopiesofchromosome21frommotherandfather.Thispaperestablishedanon-apyrase3-enzymepyrosequencingmethod,whichownsagoodperform-anceofquantitativeanalysis.ThemethodisespeciallysuitabletoallelicquantificationofanSNP,enablingtherapiddiag-nosisofDown’ssyndromebyanalyzingalleleratioofSNPsonchromosome21.Keywords:apyrase;3-enzymepyrosequencingmethod;Down’ssyndrome;SNP(singlenucleotidepolymorphism)[1,2]DNA(DNApolymerase)(ATPsulfurylase)(luciferase)(apyrase)4DNA:DNA,dNTP,dNTPDNA,DNA,(PPi),(ATPS)PPiATP,(LUC)ATP,DNA,(apyrase)dNTPATP,[3],apyraseDNAdNTP,ATP,apyraseapyrase,dNTPapyrase,PPiATP,[4];,apyrase,dNTP,,apyraseapyraseATPdNTP,DNAATP,dNTP,,ATPdNTP,dNTP,ATP,,ATP,N,[5,6]ATP[7],,(bead-LUC)ATP(bead-ATPS),apyrase,bead-LUCbead-ATPSKlenowDNA,dNTPdNTPATP,,DNA,SNP,,,,,21SNP168SNP,,11.11.1.1样品DNAEDTA;8DNA5:519,G211.1.2主要试剂TaqDNADNATaKaRa;(Luciferin)Sigma;KlenowDNApolymerase(exo-)(ATPsulfurylase)(Luciferase);(DynabeadsTM-280Streptavidin)Invitrogen1.21.2.1固相单链DNA模板的制备DNA,()PCR,PCR,(beads)[8],,1SNP-21.2.2bead-LUC和bead-ATPS的制备10μL,50μLwashingbuffer[0.1mol/LTris-AcpH7.9,0.5mmol/LEDTA,5mmol/LMg(Ac)2,0.4mg/mL(PVP),0.02%(BSA),1mmol/L(DTT)],,10μL20μLwashingbuffer,4℃1h,,50μLwashingbufferbeads3,beads10μLwashingbuffer,bead-LUC10μLATPbeads,bead-ATPS,bead-LUCbead-ATPSbead-LUC4℃1.2.3三酶焦测序体系bead-LUCbead-ATPSDNA,10μL,:0.1mol/LTris-AcpH7.9,0.5mmol/LEDTA,5mmol/LMg(Ac)2,0.4mg/mLPVP,0.02%BSA,1mmol/LDTT,4μmol/L5′-(APS),0.4mmol/Ld-,90U/mLDNAKlenow(exo-),BPCL(),dNTP,dNTP,3,,10μL1.2.4三酶焦测序体系的优化bead-LUCbead-ATPS:bead-ATPSbead-ATPSDNA,bead-LUCbead-ATPSDNA,,31.2.5三酶焦测序体系定量检测杂合型SNPPCRSNP,PCR,,SNP1μL,SNP,SNP,SNP,,3,11.2.6等位基因特异性PCR法测定SNPSNP3:,[9]表1引物序列SNP(GenBank)a(5′→3′)SNP-1(rs1042917)bio-SNP1-FWSNP1-RVSNP1-ANGTGGGAGGCGATGAGATGGGATTGATGAGGCGGTCGTAGGCACTTGAAGCTCGACAGGGAGTCGATASNP-2(rs4818219)bio-SNP2-FWSNP2-RVSNP2-ANAGGTCTCACTTGTTCAAATTCCCTGGAGCCCTTCCCACTACATCGTACAGAATTGTGTGGCTTGCGCAGTG:aFW;RV;AN520HEREDITAS(Beijing)201032PCR,PCR,SNP1.2.7数据分析3,origin7.5,22.11.2.2bead-LUCbead-ATPSbeads1.1amol⁄μL,,bead-LUCbead-ATPS2.1×105RLU/μL1.37×10−2mU⁄μL3μLbead-LUC3μLbead-ATPS1μLDNA,10μL,DNAdNTP,,1,ATTCTG14,4,,3,ATPDNA,,DNA图1三酶焦测序体系测定已知DNA序列的典型图谱2.2bead-ATPSbead-LUC,ATPATPSATP,;ATP,,,,3ATT,bead-ATPSbead-LUCbead-ATPSbead-LUC3μL,DNA1μL,bead-ATPS0.5μL1μL2μL3μL,2-(1),DNAbead-LUC,bead-ATPS,bead-ATPS2μL,,bead-ATPS2μL,2μLbead-ATPSbead-LUC,bead-ATPS2μL,DNA1μL,bead-LUC0.5μL1μL2μL3μL,2-(2),DNAbead-ATPS,bead-LUC,,2μL(2-(2)CD),,2μL,,2.3SNP,,,21SNP1:1,;,21,SNP1:22:181621SNP,rs1042917rs4818219,,0.5120.321[9],3′SNPSNP,5:521图2三酶焦测序体系中bead-ATPS和bead-LUC用量的优化(1):ABCDbead-ATPS0.5μL1μL2μL3μL;(2):ABCDbead-LUC0.5μL1μL2μL3μL,DNAA-T-TdNTP3243AB(rs1042917)FG(rs4818219)SNP,,21;3C(rs1042917)H(rs4818219)SNP,,21,21;3DE(rs1042917)IJ(rs4818219)SNP,,,,21,21,2:1,21,,PCRSNP(168):SNP,PCR21,43图3不同样本在rs1042917位点(A、B、C、D、E)和rs4818219位点(F、G、H、I、J)处的典型三酶焦测序体系测定图谱522HEREDITAS(Beijing)201032图4图3中各样