07HLA分型

PCR-SSP检测HLA-B27一、实验目的掌握PCR-SSP的原理，了解HLA基因分型的基本方法。二、实验原理编码HLA的基因具有高度的多态性，每一个基因座位上有众多的复等位基因，而每一个等位基因都有其各自的DNA序列，因此可用相应的序列特异性引物（sequencespecificprimers，SSP）进行扩增。通过控制PCR反应条件，特异性引物仅扩增与其相应的等位基因，而不扩增其他的等位基因。[e-mail]2/541322m2121AllelicpolymorphismisconcentratedinthepeptideantigenbindingsitePolymorphismintheMHCaffectspeptideantigenbindingAllelicvariantsmaydifferby20aminoacidsClassII(HLA-DR)[e-mail]5/54MolecularbasisofMHCtypesandvariantsPOLYMORPHISMVariation1%atasinglegeneticlocusinapopulationofindividualsMHCgenesarethemostpolymorphicknownPOLYGENISMSeveralMHCclassIandclassIIgenesencodingdifferentt[e-mail]6/54SimplifiedmapoftheHLAregionMaximumof9typesofantigenpresentingmoleculeallowinteractionwithawiderangeofpeptides.ClassIIIMHCClassIIDPLMP/TAPDMDQDR1BCAMHCClassIPolygenyCLASSI:3typesHLA-A,HLA-B,HLA-C(sometimescalledclassIagenes)CLASSII:3typesHLA-DPHLA-DQHLA-DR.4533extraDR[e-mail]7/54PolymorphismintheMHCVariation1%atasinglegeneticlocusinapopulationofindividualsEachpolymorphicvariantiscalledanalleleInthehumanpopulation,over1,200MHCalleleshavebeenidentified2317[e-mail]8/54Example:IfMHCXwastheonlytypeofMHCmoleculePopulationthreatenedwithextinctio[e-mail]9/54Example:IfeachindividualcouldmaketwoMHCmolecules,MHCXandYImpactontheindividualdependsupongenotypePathogenthatevadesMHCXMHCX[e-mail]10/54Example:IfeachindividualcouldmaketwoMHCmolecules,MHCXandY……andthepathogenmutatesPopulationthreatenedwithextinctionSurvivalofindividualthreatenedPathogenthatevadesMHCXbuthassequencesthatbindtoMHCYMHCXXMHCXYMHCYYThenumberoftypesofMHCmoleculecannotbeincreasedadinfinitum…[e-mail]11/54YRYRXYXXXXRXYRYXRYYRYYXRXRXRYRFrom2MHCtypesand2variants…….10differentgenotypesVariantsofeachtypeofMHCmoleculeincreasetheresistanceofthepopulationfromrapidlymutatingornewlyencounteredpathogenswithoutincreasingthenumberoftypesofMHCmoleculeVariantMHCmoleculesprotectthepopulationPathogenthatevadesMHCXandYMHCXYMHCXXMHCYYMHCXXRMHCYYR…[e-mail]12/54BCADPDQDR1PolygenyBCADPDQDR1VariantallelespolymorphismGenesintheMHCaretightlyLINKEDandusuallyinheritedinagroupThecombinationofallelesonachromosomeisanMHCHAPLOTYPEBCADPDQDR1AdditionalsetofvariantallelesonsecondchromosomeMHCmoleculesareCODOMINANTLYexpressedTwoofeachofthesixtypesofMHCmoleculeareexpressed[e-mail]13/54InheritanceofMHChaplotypesBCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRXParentsDP-1,2DQ-3,4DR-5,6B-7,8C-9,10A-11,12DP-9,8DQ-7,6DR-5,4B-3,2C-1,8A-9,10DP-1,8DQ-3,6DR-5,4B-7,2C-9,8A-11,10DP-1,9DQ-3,7DR-5,5B-7,3C-9,1A-11,9DP-2,8DQ-4,6DR-6,4B-8,2C-10,8A-12,10DP-2,9DQ-4,7DR-6,5B-8,3C-10,10A-12,9BCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRBCADPDQDRChildrenPCR-SSP检测HLA-B27一、实验目的掌握PCR-SSP的原理，了解HLA基因分型的基本方法。二、实验原理编码HLA的基因具有高度的多态性，每一个基因座位上有众多的复等位基因，而每一个等位基因都有其各自的DNA序列，因此可用相应的序列特异性引物（sequencespecificprimers，SSP）进行扩增。通过控制PCR反应条件，特异性引物仅扩增与其相应的等位基因，而不扩增其他的等位基因。[e-mail]18/541322m2121AllelicpolymorphismisconcentratedinthepeptideantigenbindingsitePolymorphismintheMHCaffectspeptideantigenbindingAllelicvariantsmaydifferby20aminoacidsClassII(HLA-DR)ClassIEveryHLAallelehasitsownuniquesequencesSpecificsequenceprimer(SSP)isdesignedtobindtotheallelicsequencescomplementarilySequencespecificprimerisusedtotypeHLAallelewithPCRSequencespecificprimerisusedtotypeHLAallelewithPCR三、实验材料和方法1、血样：0.2mlEDTA抗凝血2、红细胞裂解液3、白细胞裂解液4、PCR混合液PCRbufferHLA-B27SSPTaqdNTPinternalpositivereferenceprimersetcHLA-B27检测操作流程一、DNA的提取1、取1mlRBC裂解液入血样管中混匀，裂解RBC;2、离心4000rpm2min;3、重复步骤1-2两次，最后用棉签吸干管壁液体；4、取50ulWBC裂解液入上述WBC管中混匀；5、将WBC管于60℃水浴消化20min；6、取出WBC管再于100℃，3-5min，灭活蛋白酶K；7、离心10000rpm2min。上清即为富含DNA的PCR模板。二、PCR扩增1、吸2ul模板DNA入装有PCR混合液的小管中；2、将小管置于PCR仪内进行扩增。（需80min）×12×2394oC2min94oC12sec65oC1min94oC12sec61oC50sec72oC30sec37oC10secHLA-B27扩增程序三、电泳检测•吸PCR产物10ul加到2%琼脂糖凝胶孔内；•将琼脂糖凝胶板置电泳槽内电泳160V20min后取出，观察结果。四、结果观察SpecificbandscanbeseenwithHLA-B27(+)samplesHLA分型方法概述•血清学分型—微量淋巴细胞毒实验•细胞学分型—混合淋巴细胞培养•基因分型–PCR-RFLP–PCR-SSCP–PCR-SSP–PCR-SSOP–RSCA–PCR-SBT