八角RAPD-PCR反应体系的建立及引物筛选研究

Establishment of RAPD-PCR Reaction System and Primer Selection

在提取高质量八角基因组DNA的基础上,通过对其RAPD反应体系的Taq DNA聚合酶、dNTPs浓度、Mg2+浓度、引物浓度等因子的优化,建立了稳定、重复性高的八角RAPD-PCR反应体系。研究结果表明,在25μL PCR反应体系中,含1.0 UTaq DNA聚合酶,0.15 mmol.L-1 dNTPs,2.0 mmol.L-1 Mg2+,2.0μmol.L-1引物,20～80 ng模板。最佳反应程序为,94℃预变性5 min,扩增后94℃变性30 s,31～37℃退火30 s,72℃延伸30 s,35个循环;72℃完全延伸7 min,4℃保存。在建立RAPD-PCR反应体系的基础上,从240条RAPD引物中筛选出15条扩增条带清晰、稳定性好的引物,并通过各引物的退火温度梯度实验,确定了各引物的最适退火温度。

The RAPD - PCR reaction system of Illicium verum was established through optimizing a series of factors including Taq, the concentration of dNTPs, the concentration of Mg^2＋ and the concentration of primers. The results indicated that the optimized reaction system of 25 μL was 1.0 U Taq polymerase, 0. 15 mmol · L^-1 dNTPs, 2.0μmol · L^-1 primer, 20 -80 ng template DNA. The proper PCR reaction conditions were as follows： predenaturing at 94 ℃ for 5 min, followed by 35 cycles of denaturing at 94 ℃ for 30 s, annealing at 37 ℃ for 30 s, extension at 72 ℃ for 30 s, and final extension at 72℃ for 7 min. Using the established reaction system, fifteen stable primers with clear band were screened out from 240 tested primers, and the optimum annealing temperatures for different primers were developed.