摘要
以国兰杂交品种(系)为材料,采用L16(45)正交试验设计对Taq DNA聚合酶、Mg2+、dNTPs、模板及引物等5个重要影响因素进行优化,确定国兰杂交品种(系)SRAP-PCR反应的最优反应体系为(25μL体系):Taq DNA聚合酶,1 U;Mg2+,1.8 mmol/L;dNTPs,0.08 mmol/L;模板DNA,100 ng;引物,上下游各0.3 mmol/L;10×PCR Buffer,2.5μL。此外,本试验采用DNA混合池技术(DNA pooling)快速筛选SRAP引物,从180对SRAP引物组合中筛选出39对扩增产物丰富、多态性高的引物组合。与常规方法相比,该技术明显缩短试验周期、节约了模板用量,为快速高效筛选SRAP引物提供了新思路。
The SRAP-PCR system of the hybridization cuhivars （Lines） of Chinese Cymbidium was optimized by the orthogonal experiment which was in four levels of five factors （Taq DNA polymerase, Mg2＋, dNTPs, DNA template and Primer）. The results showed that the optimized PCR reaction system containing Taq DNA polymerase 1 U, Mg2＋ 1.8 mmol/L, dNTPs 0.08 mmol/L, DNA template 100 ng, each primer 0.3 mmol/L and 10xPCR buffer 2.5 uL. By using DNA pooling technology, 39 primer combinations were quick screened among 180 primer combinations, which had abundant polymorphism bands. Comparing with conventional screening method, the DNA pooling method can greatly shorten experimental period and significantly reduce the consumption of DNA template. And this showed a new way for quickly screening SRAP primers.
出处
《热带作物学报》
CSCD
北大核心
2014年第5期925-932,共8页
Chinese Journal of Tropical Crops
基金
国家科技支撑计划项目(No.2007BAD07B01)
