胡枝子属ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction
System for Lespedeza spp.

摘要: 在利用ISSR技术对胡枝子属种质资源遗传多样性进行研究的实验过程中,对影响PCR扩增效果的一些因素如DNA的提取、模板DNA质量浓度、Taq酶用量、引物用量、dNTP的用量以及退火温度等指标进行筛选和优化,筛选优化出可用于胡枝子属ISSR- PCR分析较适宜的PCR反应条件:Taq酶1.0 U,2 μL的10×Buffer (200 mM Tris-HCl;200 mM KCl;100 mM (NH₄)₂SO₄;15 mM MgCl₂),模板DNA 40 ng,dNTP 0.2 m mol/L,引物0.2 μmol/L.

Abstract: The influencing factors including genomic DNA extraction, concentration of template DNA, dosage of Taq DNA polymerase, primer concentration, dNTP dosage and the annealing temperature upon the amplification effect with PCR technique were studied for optimizing the ISSR reaction conditions for studies on the genetic diversity of Lespedeza spp.germplasm resources. The results showed that the optimum reaction conditions for ISSR PCR of Lespedeza spp. were as follows:1.0 U Taq DNA(produced by TIANWEI company)+2 μL 10×Taq buffer (200 mM Tris-HCl; 100 mM(NH₄)₂SO₄;200 mM KCl;15 mM MgCl₂)+40 ng template DNA+0.2 m mol/L L dNTP+0.2 μmol/L primer.