温郁金RAPD-PCR反应体系建立及条件优化

Optimization of RAPD Reaction systems for Curcuma wenyujin

在提取纯化温郁金DNA的基础上,利用PCR扩增技术,对Mg2+、dNTP、模板DNA、引物、Taq聚合酶等反应条件进行优化,建立温郁金基因组DNA的RAPD-PCR最佳反应体系。结果表明:最佳条件是总体积为25μL,其中Mg2+(25mM)2μL,Taq聚合酶(5U/μL)0.3μL,引物浓度(20μM)1.0μL,模板DNA(5ng/μL)1.0μL,dNTP(2.5mM)2.0μL,10×PCRbuffer2.5μL;PCR扩增程序为94℃预变性5min;94℃变性35s,36℃退火1min,72℃复性1.5min,共42个循环;最后72℃延伸10min,该研究得出的体系是温郁金RAPD-PCR的最适宜反应体系,具有省时、经济、简便以及扩增条带较清晰、稳定等特点,为今后温郁金的遗传多样性研究奠定了基础。

Some reaction condition was optimized by amplifying technology and method of PCR based on isolating genomic DNA from Curcurna wenyujin, to establish the best reaction condition of RAPD-PCR amplification system of Curcurna wenyujin. Results showed that the best amplification system for Curcurna wenyujin was as follows, total reaction volume 25/IL, including Mg^2＋ （25 raM）2. 0 μL, Taq polymerase（5 U/L）0. 3μL, random primer （20 M）1. 0μL,template DNA 1. 0 μL, dNTP（2. 5 raM）2. 0 μL, 10×PCR buffer 2. 5 μL; the amplification procedure conditions were pre-denature at 94℃ for 5 rain followed by denature at 94℃ for 35 s,armealing at 36℃ for 1 rain, extension at 72℃ for1.5 rain, cycling 42 times, last extension at 72℃ for 10 rain. This amplification system was very economical, convenient and the band was clear and stable. It was the most appropriate system, based for the genetic diversity of curcurna wenyujin.