HLA-B27检测.ppt

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,PCR-SSP,检测,HLA-B27,一、实验目的,掌握,HLA,基因分型的原理，学习,PCR-SSP,基因分型方法。,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（,sequence specific primers,，,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。,1,实验步骤,PCR,扩增,琼脂糖凝胶电泳,抽提,DNA,2,PCR,扩增的基本原理,模板,DNA,的变性（热变性）,模板,DNA,双链或经,PCR,扩增形成的双链,DNA,解离,模板,DNA,与引 物的退火,(,复性,),：,模板,DNA,经加热变性成单链后，温度降至,55,左右，引物与模板,DNA,单链的互补序列配对结合；,引物的延伸：,DNA,模板,-,引物结合物在,TaqDNA,聚合酶的作用下，以,dNTP,为反应原料，靶序列为模板，按碱基配对与半保留复制原理，合成一条新的与模板,DNA,链互补的半保留复制链,重复循环变性,-,退火,-,延伸三过程，,2,3,小时就能将待扩目的基因扩增放大几百万倍。,3,PCR,扩增体系,模版,DNA,上游引物,下游引物,dNTP,（原料）,缓冲体系,(,各种离子与水,),耐热的,DNA,聚合酶,4,Polymorphism in the MHC,Variation 1%at a single genetic locus in a population of individuals,Each polymorphic variant is called an allele,In the human population,over 1,200 MHC alleles have been identified.MHC genes are the most polymorphic known,5,Polymorphic residues are located in the antigen-binding groove,Variation in amino acid sequences changes the shape of the groove,6,Schematic diagram of class I and class II MHC genes,mRNA transcripts,and protein molecules,7,Every HLA allele has its own unique sequences,8,Specific sequence primer(SSP),is designed to bind to the allelic sequences complementarily,9,Sequence specific primer is used to type HLA allele with PCR,10,Sequence specific primer is used to type HLA allele with PCR,11,三、实验材料和方法,1,、血样：,0.2 ml EDTA,抗凝血,2,、红细胞裂解液,3,、白细胞裂解液,4,、,PCR,混合液,PCR buffer,HLA-B27 SSP,Taq,dNTP,internal positive reference primers,12,HLA-B27,检测操作流程,一、,DNA,的提取,1,、取,1ml RBC,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,100ulWBC,裂解液入上述,WBC,管中混匀；,5,、将,WBC,管于,60,o,C,水浴消化,20min,；,6,、取出,WBC,管再于,100,o,C,，,3-5min,，灭活蛋白酶,K,；,7,、离心,10000,rpm,2min,。上清即为富含,DNA,的,PCR,模板。,13,二、,PCR,扩增,1,、吸,2ul,模板,DNA,入装有,PCR,混合液的小管中（注意不要加到石蜡油层）；,2,、将小管置于,PCR,仪内进行扩增。（需,80min,）,12,23,预变性：,94,o,C 2min,变性：,94,o,C,12sec,复性：,62,1min,延伸：,72,30sec,变性：,94,o,C 12sec,复性：,58,o,C 50sec,延伸：,72,o,C,30sec,37,o,C 10sec,HLA-B27,扩增程序,14,三、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。,15,四、结果观察,16,Specific bands can be seen with HLA-B27(+)samples,17,HLA,分型概述,方法比较,血清学分型,细胞学分型,基因分型,应用,移植配型,疾病相关性研究,法医学,18,