降落-重叠PCR法四重融合构建平菇同源重组片段

Quadruple DNA Fragments Fusion using Touchdown-Overlap Extension PCR

对多个长片段的基因融合目前仍缺少有效的方法.本文提出一种新的融合PCR策略,即在常规的重叠PCR的第1步和第2步均增加1个降落PCR程序,减少不适当的退火温度和PCR产物3'端额外碱基A对片段融合、扩增的影响,提高正确融合与扩增的效率.结果表明,为构建平菇葡聚糖合成酶启动子的同源重组序列,在4个长度分别是1 015 bp、2 822 bp、2 206 bp和1 008 bp的片段进行融合时,在重叠PCR的第1步加上退火温度61.5℃～57.5℃、每降落0.5℃进行1个循环的降落PCR程序,在重叠PCR的第2步加上退火温度60℃～56℃、每降落0.5℃进行1个循环的降落PCR程序,经过1次PCR即获得顺序正确的全长融合片段.测序结果与4个片段序列的一致性达到98.5%,降落-重叠PCR法对多个长片段的基因融合具有较高的应用价值.

Here we introduced a genes fusion procedure using touchdown PCR （TD PCR） in the step I and II of classic overlap extension PCR （OE PCR） to reduce the inappropriate annealing and the extra 3＇-end adenine which added by Tag DNA polymerase. In an attempt to construct a homologous recombination fragment to replace the glucan synthase promoter in Pleurotus ostreatus, four fragments of 1 015, 2 822, 2 206 and 1 008 bp in length were used for the fragment fusion. In step I, the annealing temperature were decreased by 0. 5 ℃ from 61.5 ℃ to a touchdown at 57.5 ℃starting from the second cycle. In step II, the annealing temperature were decreased by 0.5 ℃ from 60 ℃to a touchdown at 56 ℃ with the supply of LA DNA polymerase. The desired fusion product of 7.05 kb was obtained through the overlap PCR with one round, and then cloned into a T-vector and sequenced. A 98.5% consensus between the fusion product and the four separate DNA fragments was detected. The results suggested that TD-OE PCR could be an effective method to prepare long multiple fragment fusions for the research in somatic cell knockout/in or partial fungal genome synthetic applications.