黑龙江林蛙ISSR反应体系的建立与优化

以黑龙江林蛙为材料,使用引物ISSR807,采用正交优化方法对影响PCR反应的Mg2+、dNTPs、引物、DNA聚合酶、模板进行体系优化,对引物的最佳退火温度进行选择,建立适合黑龙江林蛙的ISSR-PCR分析的最佳体系:在25μL总体积中,包含10×PCR Buffer 2.5μL,Mg2+4.0 mmol.L-1,dNTPs 0.15 mmol.L-1,引物0.3μmol.L-1,TaqDNA聚合酶0.5 U,DNA模板2.0 ng.μL-1,最佳退火温度53.8℃。为利用该技术对黑龙江林蛙遗传多样性分析和构建遗传图谱提供一定的依据。

Establishment and optimization of ISSR reaction system for Rana amurensis Boulenger

Rana amurensis Boulenger was chosen to study the affecting factors of ISSR-PCR amplification.Orthogonal design of five factors(Mg2 +,dNTPs,primer,TaqDNA polymerase,and tem-plate) at four levels was used to establish the suitable ISSR-PCR system by primer ISSR807 in Rana amurensis Boulenger,and the suitable anneal temperature was yielded from gradient PCR on temperature.The results showed that the optimized ISSR-PCR system(25 μL reaction volume) in Rana amurensis Boulenger was proved to be: 10×PCR buffer 2.5 μL,Mg2 + 4.0 mmol·L-1,dNTPs 0.15 mmol·L-1,primer 0.3 μmol·L-1,Taq DNA polymerase 0.5 U,DNA template 2.0 ng·μL-1;and suitable anneal temperature was determined to be 53.8 ℃.The results lay a foundation for the genetic diversity analysis and genetic map construction of Rana amurensis Boulenger.