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研究了两轮多重反转录巢式PCR扩增全长基因的方法。先用多重巢式引物和cDNA进行第1轮PCR扩增;再用纯化的第1轮PCR产物和相同的引物进行第2轮扩增;最后通过改变Mg2+浓度、退火时间和退火温度进行PCR条件的优化,以增加PCR扩增的产量,用于克隆。结果显示该方法方便和快速地扩增出了草莓(Fragariaananassacv.SweatCharlie)高产量和高质量的全长GalUR基因,也非常快速地扩增出了用于基因构建的带有酶切位点的全长GalUR基因。该方法也可以用于其它全长基因的克隆。
Two rounds of multiplex reverse transcription nested PCR amplification of full-length gene. The first round of PCR amplification was performed with multiple nested primers and cDNA. The first round of PCR with the purified product and the same primer were used for the second round of amplification. Finally, the PCR conditions were changed by changing Mg2 + concentration, annealing time and annealing temperature Optimization to increase the yield of PCR amplification for cloning. The results showed that this method was convenient and rapid amplification of the strawberry (Fragariaananassacv.SweatCharlie) high yield and high quality full-length GalUR gene, but also very quickly amplified for gene construction with enzyme cutting sites Long GalUR gene. This method can also be used for the cloning of other full-length genes.