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针对RAPD技术重复性不理想的问题,以3个文蛤群体为研究材料,确立“两步法”建立高重复性RAPD优化体系。第一步初筛,即以正交分析法对文蛤RAPD反应体系中各关键组分(DNA模板、引物、dNTPs、Mg2+、Taq聚合酶),均设4个水平进行分析,确定两个待选体系;第二步检测,即以本研究中创立的重复性检测方法对待选方案进一步筛选。结果表明:初筛的两个反应条件体系Ⅰ和体系Ⅱ尽管在扩增条带的产量方面无明显差异,但体系Ⅱ各重复性检测指数明显高于体系Ⅰ;体系Ⅱ为更适合文蛤的RAPD高重复性反应体系。“两步法”中采用的多因素联合优化方案和RAPD重复性检测方法对RAPD技术的改善具有一定的指导意义。
Aiming at the problem of unsatisfactory repeatability of RAPD technique, three clam groups were selected as research materials to establish a “two-step method” to establish a high-repetitive RAPD optimization system. The first step of screening, namely orthogonal analysis of RAPD reaction system of the text of the key components (DNA templates, primers, dNTPs, Mg2, Taq polymerase), were set at four levels to determine the two to be selected System; the second step of testing, that is, in this study, the repeatability of detection methods to be selected screening plan further screening. The results showed that although the two reaction conditions system I and II did not show significant difference in the yield of amplified bands, the repeatability indexes of system Ⅱ were significantly higher than that of system Ⅰ. System Ⅱ was more suitable for RAPD High repetitive reaction system. The two-step method used in the multi-factor optimization program and RAPD repeatability detection methods for RAPD technology to improve some guidance.