摘要
以转基因旱稻为材料,对Tail-PCR方法进行了分析优化,包括DNA样品质量、扩增产物稀释程度、酶的种类、简并引物组合方式、引物结合偏好性等。结果表明,DNA样品质量的高低是影响试验成功的重要因素,样品纯度不高导致扩增无法正常进行;扩增后对产物稀释不充分会影响下轮扩增效果,适当增大产物稀释倍数可在下轮扩增中增大出带可能;LA Taq酶与普通Taq酶相比扩增效果没有明显不同,使用普通Taq酶即可进行Tail-PCR扩增;混合简并引物在旱稻中并没有获得更好的扩增效果。此外,发现简并引物对于不同物种存在偏好性,常规4种简并引物与旱稻基因组均可高效结合。优化后的Tail-PCR技术结果稳定,重复性好,可以准确地获得转基因旱稻插入位点的旁侧序列。
Tail-PCR about transgenic upland rice was analyzed and optimized,including the quality of DNA samples,dilution degree of amplification products,enzyme types,degenerate primer combinations,primer preferences,etc. The results showed that the quality of DNA sample was an important factor for the success of the experiment. Low-purity sample led to an abortive amplification. The result of the next round amplification was affected by the dilution of the product of the previous round,and appropriately increasing product diluted multiples could increase the belts in the next round of amplification. The effect of LA Taq enzyme in this experiment was not significantly different from that of ordinary Taq enzyme,therefore the normal Taq enzyme can be used to amplify effectively. The effect of amplification with mixed degenerate primers in upland rice was not better than that of separate amplification. Moreover,it was found that degenerate primers had preference in different species. The conventional four kinds of degenerate primers could all effectively combine with the genome of the upland rice. The optimized Tail-PCR about upland rice in our studies is of stability and good repeatability. It can be accurate to obtain flanking sequence of insertion site in transgenic upland rice.
出处
《河南农业科学》
CSCD
北大核心
2018年第1期7-11,共5页
Journal of Henan Agricultural Sciences
基金
国家转基因生物新品种培育科技重大专项(2016ZX08001003-006)
河北科技大学五大平台开放基金课题(SW21
2015PT51)
河北省高等学校科学技术研究项目(QN2017071)
关键词
旱稻
Tail-PCR技术
优化
影响因素
Upland r ice
Technology of Tail- PCR
Optimizing
Influence factors
