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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.
At present, there is still no effective method for gene fusion of multiple long fragments.This paper presents a new fusion PCR strategy, which includes adding one drop PCR program in step 1 and step 2 of conventional overlap PCR to reduce inappropriate , And the effect of extra base A at the 3 ’end of the PCR product on the fusion and amplification of the fragments, so as to improve the efficiency of correct fusion and amplification. The results showed that in order to construct the homologous recombination sequence of the Pleurotus ostreatus glucan synthase promoter, , The four annealing lengths of 1 015 bp, 2 822 bp, 2 206 bp and 1 008 bp were used for the fusion. The annealing temperature was 61.5 ° C-57.5 ° C and 0.5 ° C for each step The 1-cycle descending PCR program was followed by one cycle of the descending PCR program at step 2 of the overlap PCR plus an annealing temperature of 60 ° C. to 56 ° C. with a drop of 0.5 ° C. After one PCR, sequential full-length fusions were obtained Fragment.The sequencing result was 98.5% identical to the four fragment sequences, and the lap-overlap PCR method was of high value for gene fusion of multiple long fragments.