小麦随机扩增多态性DNA的反应体系探索

Exploration of reaction system of randomly amplified polymorphic DNA analysis in wheat

随机扩增多态性DNA(RAPD)是近年来发展起来的一种DNA多态性检测技术。通过以小麦基因组DNA为模板 ,对RAPD反应程序中的一些重要参数进行摸索和优化试验 ,建立了随机扩增多态性DNA分析应用反应体系。即反应体积为 2 5μ1,Mg2 + 浓度为 2 0mM ,dNTP浓度为 150 μM ,模板DNA量 5ng～2 5ng ,随机引物量 30ng～ 4 5ng ,Tap酶 1单位 ;反应过程为 90℃变性 30s ,38℃结合 1min ,72℃延伸 2min(最后一个循环 10min) ,共 4 0个循环。该体系可有效地应用在小麦遗传育种研究中 ,重复性好 ,可靠性高。

Randomly amplified polymorphic DNA (RAPD) is an advanced technique used for detection of the DNA polymorphisms developed in recent years. Based on the genomic DNA extracted from wheat, some essential factors that might affect the result of RAPD assay was tested and compared. An optimal reaction system suitable for the assay and usage of RAPD in wheat was established. Amplification reactions contained 2 0mM Mg 2+ ,150μM of each dNTP, 30ng～45ng decamer primer, 5ng～25ng genomic DNA and 1 unit Taq DNA polymerase in a volume of 25μ1. The reaction program was devised for 40 cycles, each with 30 sec at 90℃, 1 min at 38℃, 2 min at 72℃, and 10 minutes at 72℃ in the final extension. This system with good reproducibility and high reliability could be effectively used for RAPD analysis in the program of wheat genetics and breeding.