缢蛏SRAP-PCR体系的正交优化  

Optimization of SRAP-PCR System with Orthogonal Design in Sinonovacula constricat

作　　者：钟玉民[1] 刘达博[1] 冯冰冰[1] 陈慧 林国文 李家乐[1] 

机构地区：[1]上海海洋大学水产与生命学院农业部水产种质资源与养殖生态重点开放实验室,上海201306 [2]福建省闽东水产研究所,宁德352100

出　　处：《生物技术通报》2011年第6期99-103,共5页Biotechnology Bulletin

基　　金：国家"863"计划项目(2006AA10A410);福建省海洋与渔业局重点项目(闽海鱼2007015)

摘　　要：运用L_(16)(4~5)正交设计对影响缢蛏SRAP-PCR反应的5个因素:Taq酶浓度、Mg^(2+)浓度、模板DNA浓度、dNTPs浓度和引物浓度在4个水平上进行了优化试验,PCR结果采用SPSS v16.0软件分析。结果表明,各因素对SRAP-PCR反应的影响依次为:引物>Taq酶>模板DNA>Mg^(2+);缢蛏SRAP反应最佳体系为:在20μL PCR反应体系中,引物0.3μmol/L、Taq酶0.5 U、模板DNA 50 ng、dNTPs 0.2 mmol/L及Mg^(2+)2 mmol/L。用不同缢蛏的基因组DNA两次SRAP-PCR扩增,8对引物均能扩增出清晰且重复性好的谱带。因而建立的缢蛏反应体系稳定可靠。The orthogonal design was used to optimize SRAP-PCR amplification system of Sinonovacula constricat on four levels, the experiment was performed on five factors ： the concentration of Taq DNA polymerase, Mg2＋ , DNA template, dNTPs and Primer. The results of PCR were analyzed by SPSSv16. 0. The results showed：the effective degree of the factors on the SRAP-PCR reaction was in the order：Primer 〉 Taq DNA polymerase 〉 DNA template 〉 dNTPs 〉 Mg2＋ ;the most suitable SRAP-PCR system for S. constricat, was established ： the 20μL volume system contained ：0. 3 μmol/L Primer,0. 5 U Taq DNA polymerase,50 ng DNA template, 0. 2 mmol/L dNTPs and 2 mmol/L Mg2＋. In this two SRAP-PCR amplificaations with different S. constricat template, eight primers could amplify the clear and stable bands. So the established system of SRAP in S. constricat is stable.

关 键 词：缢蛏 SRAP 正交设计 优化 

分 类 号：S917.4[农业科学—水产科学]