长心卡帕藻RAPD-PCR反应体系的正交优化研究

Study on Optimization of RAPD-PCR Reaction System for Kappaphycus alvarezii by Orthogonal Design

采用SD(S十二烷基硫酸钠,20%)法提取了大型海藻长心卡帕藻(Kappaphycus alvarezii)基因组DNA。通过单因子梯度试验,确定了影响随机扩增DNA多态性(RAPD)扩增结果的模板、Mg2+、dNTPs、S22引物、Taq的适宜浓度、退火温度和反应的最佳循环次数;利用正交试验优化了模板、Mg2+、dNTPs、S22引物、Taq的配比浓度。结果表明,在进行长心卡帕藻的RAPD扩增时,在总体积为25μl的反应体系中,模板、Mg2+、dNTPs、S22引物、Taq的最佳浓度分别为15ng、2.0mmol/L、0.2mmol/L、0.25μmol/L、2.5U;退火温度为37℃。反应程序为94℃预变性5min,然后经94℃变性30s、37℃退火1min,72℃延伸2min,进行30次循环,最后在72℃再延伸10min。

The genomic DNA of the seaweed Kappaphycus alvarezii was extracted by SDS （20%）method. The template,Mg^2＋,dNTPs,S22 primer, Taq concentrations,and annealing temperature and the best cycle times were optimized for RAPD-PCR reaction system in K. alvarezii with single-factor and orthogonal design experiment. In 25μ1 RAPD- PCR reaction system of K. alvarezii,the most suitable concentration of template,Mg^2＋,dNTPs,S22 primer,Taq polymerase were 15ng,2.0mmol/L,0.2mmol/L,0.25μmol/L and 2.5U,respectively. And the PCR program for RAPD was 5min initial denaturation at 94℃,then followed by 30 cycles of 30 seconds at 94℃ （denaturation）,60 seconds at 37℃ （annealing）,120 seconds at 72℃（extension）,and a final 10 minutes extension at 72℃.