水曲柳SRAP分子标记反应系统的优化

以水曲柳叶片DNA为模板,利用SRAP(相关序列多态性)技术及L_(16)(4~5)正交试验和单因子试验方法,分析了不同浓度的DNA模板、Mg~(2+)、dNTP、引物、TaqDNA聚合酶对扩增结果的影响,最终确立的PCR最佳反应系统为:20μL体系中,2×PCR buffer、DNA模板50～100ng,Mg~(2+) 的浓度2.0mmol/L,dNTP0.15mmol/L,引物0.30μmol/L,TaqDNA聚合酶1.0U;最佳退火温度50℃.在此反应系统下,所扩增谱带清晰、稳定、多态性高.

Optimization of SRAP Reaction System in Fraxinus mandshurica

Effects of different concentrations of DNA template, Mg~(2+) , dNTP, primer, Taq DNA polymerase on amplification re-sult were studied by single factor test, orthogonal design of L_(16)(4~5) and Sequence-related amplified polymorphism marker (SRAP) technique, using the genomic DNA extracted from Fraxinus mandshurica Rupr as template. The optimal PCR system was obtained as 2 × PCR buffer, Mg~(2+) 2.0 mmol/L, dNTPs 0.15 mmol/L, primer 0. 30 μmol/L, Taq DNA poly-merase 1.0 U, and genomic DNA template 50～100 ng in 20 μL reaction system. And the optimal annealing temperature was 50 degrees C. Clear, stable and abundant polymorphic bands were obtained under the optimal reaction system.