摘要
模板DNA、引物、dNTPs、Mg2+的浓度,Taq DNA聚合酶的用量以及退火温度是影响简单序列重复区间扩增(ISSR-PCR)结果的主要因素.以桉树叶片基因组DNA为试材,系统地测试了这6个因素对桉树ISSR反应结果的影响.结果表明:最优化的反应体系即20μL反应体系中,含20 ng模板DNA、0.4μmol.L-1随机引物、0.15 mmol.L-1dNTPs、2.0 mmol.L-1Mg2+、1.25 U TaqDNA聚合酶.最佳退火温度为55℃;PCR反应程序为94℃预变性5 min;94℃变性45 s,55℃退火45 s,72℃延伸1.5 min,35个循环;72℃再延伸7 min.
Template DNA, primers, dNTPs, Mg^2＋ concentration, dose of Taq DNA polymerase and annealing temperature are primary factors to ISSR-PCR. With the genomic DNA of Eucalyptus leaves as material, the six factors were systematically analyzed. The result shows that the optimal reaction system of ISSR is the 20μL system containing 20 ng template DNA, 0.4 μmol· L^-1 random primers, 0. 15 mmol· L^-1 dNTPs, 2. 0 mmol·L^-1Mg^2＋, and 1. 25 U Taq DNA polymerase; and that the optimized annealing temperature is 55℃, the PCR procedure is ： pre-denaturizing at 94℃ for 5 rain, denaturizing at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1.5 min, reaction of 35 cycles and rextension at 72 ℃for 7 min.
出处
《中南林业科技大学学报》
CAS
CSCD
北大核心
2008年第1期44-48,共5页
Journal of Central South University of Forestry & Technology
基金
长沙市科技计划重点项目(K069083-22)部分研究内容
