《DNA分子标记》PPT课件.ppt

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, TRFP,2/50,RAPD,RAPD random amplified polymorphic DNA Williams et al. 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DNA,3/50,4/50,RAPD,实验流程 DNA提取 PCR扩增 产物检测 数据记录,5/50,RAPD,很难判断带的有无,背景影响严重,：带的亮度差异很大？,凝胶分辨率？,6/50,RAPD,7/50,RAPD,阴性对照出带,阳性对照 where,8/50,RAPD,反应体系 Buffer Mg2+ dNTP DNA 聚合酶 引物 DNA H2O,Mg2+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,9/50,RAPD,反应条件 94 36 72 引物退火温度太低 循环次数,10/50,RAPD,阴性对照 阳性对照 可重复性,11/50,RAPD,数据记录 0 1矩阵 显性标记,12/50,RAPD,缺陷 可重复性低 显性标记,13/50,RAPD,RAPD random amplified polymorphic DNA 10碱基 Williams et al. 1990 AP-PCR arbitrary primer PCR 20, 30碱基 Welsh et al. 1990 DAF DNA amplification fingerprinting 5, 8个碱基 Caetano-Anolles et al. 1991,14/50,随机扩增 RAPD, ISSR, 单核苷酸突变检验 SNP 限制性酶切 RFLP, PCR-RFLP, TRFP,15/50,ISSR,ISSR inter-simple sequence repeat Zietkiewicz et al. 1994 原理：随机扩增 引物：20bp左右 5-BDB(ACA)5-3,16/50,17/50,ISSR,50 mM of KCl, 1.5 mM of MgCl2, 0.25 mM of dNTPs, 2% formamide, 0.2 M of primer, 0.5 mM of spermidine, 0.8 U of Taq DNA polymerase enzyme (Banglore Genei, India) and 20 ng of DNA per 25l reaction Initial denaturation for 5 min at 94C, each cycle comprised 1-min denaturation at 94C, 45 s annealing at 49C, 2-min extension at 72C with a final extension for 5 min at 72C at the end of 45 cycles. Most of the patterns with extremely good polymorphism and useful information were often accompanied with a background smear. To reduce this smear, 2% formamide was used in the reaction. All the patterns generated were repeated at least three times in order to obtain reproducible data.,Joshi et al. 2000 Theor Appl Genet 100:1311,18/50,ISSR,19/50,ISSR,可重复性高于RAPD 随机扩增 显性标记,20/50,SNP,SNP single nucleotide polymorphisms 5 GGATCAGTACTGACTCAG 3 5 GGATCAGTAATGACTCAG 3,21/50,SNP,SNP变异来源 转换 transition 一种嘧啶置换另一种嘧啶 CT 一种嘌呤置换另一种嘌呤 AG 颠换 transversion 嘌呤与嘧啶互换，CA，AT等 缺失/插入,22/50,SNP的检测方法,23/50,SNP检测SNaPshot,24/50,SNP检测SNaPshot,单引物扩增,25/50,多重PCR: multiplex primers extension arrays,SNP检测 SNaPshot,26/50,SNP检测突变错配扩增检验,180bp,210bp,共显性标记,27/50,SNP,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,显性与共显性,180bp,210bp,RAPD 显性,SNP 共显性,29/50,花柱卷曲性的适应意义,AA Control,Cut aa,AA,AA,Aa,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,31/50,随机扩增与特异性扩增,可重复性 数据的可靠性,32/50,随机扩增 RAPD, ISSR, 单核苷酸突变检验 SNP 限制性酶切 RFLP, PCR-RFLP, TRFP,33/50,RFLP,RLFP restriction fragment length polymorphism 原理 限制性内切酶消化获得的DNA片段大小的变异,780bp,470bp,310bp,34/50,RFLP研究的发展,基因组DNA,酶切对象,PCR产物,末端荧光标记的PCR产物,RFLP,PCR-RFLP,TRFP,35/50,RFLP,实验流程 DNA提取 限制性内切酶消化 电泳分离 放射自显影检测,36/50,RFLP,限制性内切酶的选择 已经分离鉴定了数千种 近千种已用于商业流通 ,37/50,38/50,RFLP,放射自显影检测 为什么要采用放射自显影检测？ Southern 杂交 探针？,39/50,RFLP patterns in P. densata,40/50,RFLP,显性标记 or 共显性标记,41/50,PCR-RFLP,实验流程 DNA提取 目的片段扩增 （1 3 kb, 30kb） 限制性内切酶消化 电泳分离 EB染色或银染,42/50,PCR-RFLP,目的片段扩增 长度 Barnes 1994 PNAS 来源 nrDNA, cpDNA, or mtDNA 酶切位点,43/50,PCR-RFLP,44/50,Ge et al. 2001. Genome 44: 1136-1142,Identification of rice genomes by PCR-RFLP of Adh genes,45/50,TRFP Terminal restriction fragment patterns,实验流程 DNA提取 目的片段扩增 （1 3 kb, 30kb） 引物末端荧光标记， FAM 限制性内切酶消化 毛细管电泳分离 测序仪自动检测分析,46/50,TRFP,47/50,Ordered data vs. Unordered data,Ordered data：能够从现有基因型或者haplotype (多位点基因型)推测祖先型的数据 cpDNA PCR-RFLP,S1,S2,S3,48/50,Ordered data vs. Unordered data,Ordered data DNA sequences data,S1: 5 GGATCATGTCTGACAGCTACTGGTAATG 3 S2: 5 GGATTATGTCTGACAGCTACTGGTAATG 3 S3: 5 GGATTATG