一种高特异性的改良降落PCR.PDFVIP

6 4 生命科学研究 Vol.6 No.4 200212 Life Science Research Dec. 2002 PCR 张贵星, 袁保梅, 许培荣, 王建民, 薛乐勋 ( , 450052) : 为提高基因组DNA 中的基因PCR 检出的特异性, 设计了 种改良的降落PCR 程序, 并分别用Taq DNA 聚合酶及高保真Pfu DNA 聚合酶进行实验. 自盐藻Dunaliella bardawil 中提取基因组DNA 作为PCR 模板, 使用Taq DNA 聚合酶及Pfu DNA 聚合酶, 运用普通PCR 和降落PCR 程序,扩增胡萝卜素生物合成相关基因 ( cbr ) 上游启动子序列,并电泳比较PCR 扩增产物的特异性. 结果显示, 使用普通Taq 酶PCR, 普通 PCR 程序 产生200bp,500bp 和1272bp 长的三条带, 而TD-PCR 程序仅克隆出1272bp 的特异带; 利用高保真的Pfu DNA 聚合酶作PCR,在TD-PCR 泳道中仅有 1272bp 条带,而普通PCR 除了1272 bp 的特异带外,还出现 条500 bp 的非特异带. 无论使用普通Taq 酶或高保真酶Pfu,改良的降落PCR 程序均明显提高PCR 的特异性, 类似的 降落PCR 程序可望用于克隆用普通PCR 难以克隆的基因片段, 或在假阳性难以去除的情况下提高PCR 的特 异性. : 降落PCR; PCR特异性; Pfu DNA 聚合酶 :Q781 :A : 1007-7847(2002)04-0314-04 Modified Touchdown PCR as a High Efficient Approach to Improve the Specificity of PCR ZHAN u-ixing, YUAN Bao-mei, XU Pe-i rong, WAN Jian-min, XUE Le-xun ( Biotechnology L aboratory , Medical College of Zhengz hou Univ ersity , Zhengz hou 450052, H enan , China) Abstract: To improve the specificity of PCR in detection of genes in genomic DNA, a modified touchdown PCR method was designed by using the common Taq DNA polymerase and the Pfu DNA polymerase with proofreading ability. enomic DNA was extracted from Dunaliellabardawil, a unicellular andwall-less algabeing ableto propa- gate in solution of high concentration of NaCl. Two kinds of PCR using Taq and Pfu DNA polymerases were carried out, utilizing the genomic DNA mentioned above as a template, to amplify the upstream promoter sequence of the cbr ( carotene biosynthesis-related) gen