甜菜碱增强长片段PCR的扩增

摘要

聚合酶链式反应(PCR)作为一项非常成熟的技术可以用于基因组序列的扩增.普通的PCR技术只适合于短片段DNA的扩增,一般在6kb以下.对于6kb至十几kb甚至几十kb以上的DNA片段的扩增就非常困难.通过添加不同化学物质,发现甜菜碱对长片段PCR的扩增有非常有效的增强作用.通过对玉米总DNA以及质粒DNA的扩增,发现1mol/L到2.5mol/L甜菜碱对改进PCR扩增效果明显.通过添加甜菜碱,可以从玉米基因组中扩增出9kb以上的单拷贝片段,从质粒中扩增出16kb以上片段.经过试验,发现不同GC含量的引物需要使用不同浓度的甜菜碱.甜菜碱可以减少甚至消除长片段PCR中的非特异性扩增.同时,我们发现其它的添加物,如DMSO,甘油,甲酰胺对长片段PCR的作用不明显.%PCR is a powerful tool for the amplification of genetic sequences.It has been widely applied in molecular biology.It is generally used to amplify short segments(several hundreds basepairs to several kilobasepairs).It is difficult to amplify a long DNA segment.Based on the sequenced genes,it is known that most intact genes are very long.And intact gene is very important for the gene to express specially and effectively.Long PCR is a very useful tool to amplify intact genes for constructing special expression vectors.We have tried several chemicals to optimize long PCR system and found betaine was the best.Betaine,as an amino acid an alogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genomc.The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L.We could effectively amplify a 9 kb DNA segment from maize genome DNA and a 16 kb DNA segment from plasmid. It was shown that different primers and different targets(different GC content)needed different concentrations of betaine.Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals,such as DMSO,glycerin,formamide. They were no notable results in long PCR.