资源描述：
《《DNA分子标记》PPT课件》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。
1、DNA分子标记1/50随机扩增RAPD,ISSR,单核苷酸突变检验SNP限制性酶切RFLP,PCR-RFLP,TRFP2/50RAPDRAPDrandomamplifiedpolymorphicDNAWilliamsetal.1990原理：随机扩增引物：任意序列的10个碱基的寡核苷酸片段模板：未知序列的基因组DNA3/504/50RAPD实验流程DNA提取PCR扩增产物检测数据记录5/50RAPD很难判断带的有无背景影响严重Ｑ：带的亮度差异很大？凝胶分辨率？6/50RAPD7/50RAPD阴性对照出带阳性对照where8/50RAPD反应体系BufferMg2+dNTPDNA聚合酶引
2、物DNAH2OMg2+浓度较高核酸外切酶活性较低单引物，序列短纯度，浓度纯度9/50RAPD反应条件94℃36℃72℃引物退火温度太低循环次数10/50RAPD阴性对照阳性对照可重复性11/50RAPD数据记录0–1矩阵显性标记有有有有无有１１１１０１12/50RAPD缺陷可重复性低显性标记13/50RAPDRAPDrandomamplifiedpolymorphicDNA10碱基Williamsetal.1990AP-PCRarbitraryprimerPCR20,30碱基Welshetal.1990DAFDNAamplificationfingerprinting5,8个碱基Ca
3、etano-Anollesetal.199114/50随机扩增RAPD,ISSR,单核苷酸突变检验SNP限制性酶切RFLP,PCR-RFLP,TRFP15/50ISSRISSRinter-simplesequencerepeatZietkiewiczetal.1994原理：随机扩增引物：20bp左右5’-BDB(ACA)5-3’16/5017/50ISSR50mMofKCl,1.5mMofMgCl2,0.25mMofdNTPs,2%formamide,0.2μMofprimer,0.5mMofspermidine,0.8UofTaqDNApolymeraseenzyme(Banglo
4、reGenei,India)and20ngofDNAper25μlreactionInitialdenaturationfor5minat94°C,eachcyclecomprised1-mindenaturationat94°C,45sannealingat49°C,2-minextensionat72°Cwithafinalextensionfor5minat72°Cattheendof45cycles.Mostofthepatternswithextremelygoodpolymorphismandusefulinformationwereoftenaccompaniedwit
5、habackgroundsmear.Toreducethissmear,2%formamidewasusedinthereaction.Allthepatternsgeneratedwererepeatedatleastthreetimesinordertoobtainreproducibledata.Joshietal.2000TheorApplGenet100:131118/50ISSR19/50ISSR可重复性高于RAPD随机扩增显性标记20/50SNPSNPsinglenucleotidepolymorphisms5’–GGATCAGTACTGACTCAG–3’5’–GGAT
6、CAGTAATGACTCAG–3’21/50SNPSNP变异来源转换transition一种嘧啶置换另一种嘧啶C↔T一种嘌呤置换另一种嘌呤A↔G颠换transversion嘌呤与嘧啶互换，C↔A，A↔T等缺失/插入22/50SNP的检测方法23/50SNP检测－SNaPshot24/50SNP检测－SNaPshot单引物扩增25/50多重PCR: multiplexprimersextensionarraysSNP检测－SNaPshot26/50SNP检测－突变错配扩增检验180bp210bp共显性标记27/50SNP优点特异性的共显性基因有功能意义缺点引物设计困难28/50显性与共
7、显性23411112101180bp210bpRAPD显性1231AAABBBSNP共显性29/50花柱卷曲性的适应意义AAControlCutaaAAAAAa共显性标记的应用30/50显性标记的应用种群遗传多样性和遗传结构2345611011121111031000131/50随机扩增与特异性扩增可重复性——数据的可靠性有条带无条带特异性扩增扩增成功扩增不成功非特异性扩增扩增成功？32/50随机扩增RAPD,ISSR,单核苷酸突变检验SNP限制性酶切RF
