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.
Abstract
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.
关键词
基因融合 /
重叠PCR /
降落PCR /
同源重组
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Key words
gene fusion /
overlap extension PCR /
touchdown PCR /
homologous recombination
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中图分类号：
Q78
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参考文献
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脚注
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基金
河南省科技攻关项目（No.082102150048）
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