油茶ISSR反应体系建立及优化

Study on the Inter-simple Sequence Repeat Condition of Camellia oleifera

以改进的CTAB高盐法提取油茶嫩叶总DNA,进行简单重复序列间扩增(ISSR)分析,分别测试了退火温度、模板DNA浓度、M g2+浓度、dNTP s浓度、T aqDNA聚合酶用量和是否加入去离子甲酰胺对反应结果的影响.油茶ISSR分析的最适反应体系为:在20μL PCR反应体积中,含20 ng模板DNA,2.5 mm o l.L-1M gC l2,0.2 mm o l.L-1dNTP s,1UT aqDNA聚合酶,0.5pm o l.μL-1引物,1%去离子甲基酰胺.扩增程序为:94℃预变性2 m in;94℃变性30 s,52℃退火30 s,72℃延伸90 s,反应38个循环;最后在72℃延伸7 m in.

The study results indicate that the high grade genomic DNA using the method of CTAB with high salt precipitation was obtained and that the optimal PCR system for ISSR analysis was as follows： 2.5 mmol· L^-1 Mg^2＋, 0. 2 mmol · L^-1 dNTPs,1 ng DNA template, 1U Taq polymerase, 0. 5 pmol · μL^-1 random primer and 1% formamide deionized in 20 μL reaction solution. The augmentation procedure was： pre-denaturation at 94℃ for 2 min, denaturation at 94℃ for 90 s, annealing at 52C for 30 s, extension at 72℃ for 90 s, reaction of 38 cycles and extension at 72℃ for 7 min.