巢式-重叠PCR构建金属还原酶基因缺失片段

APPLICATION OF NESTED-SOE PCR TO CONSTRUCTION OF DNA FUSION FRAGMENTS FOR METALLOREDUCTASE GENE DISRUPTION

目的利用重叠PCR构建敲除烟曲霉金属还原酶基因所需的融合片段,并检测巢式PCR对重叠PCR的影响。方法比对烟曲霉金属还原酶基因上下游的同源性,在其同源性较好的区域设计引物,扩增出金属还原酶基因上下游片段。同时,利用PCR获得潮霉素抗性标记基因的两个片段。再通过巢式PCR将基因上下游片段与相应的标记基因片段分别连接,获得转化所需的融合片段。获得的融合片段用凝胶电泳和测序进行鉴定。结果获得的融合片段经凝胶电泳检测大小正确。经测序鉴定证实融合片段正确连接并与理论序列有很高的一致性。与常规重叠PCR相比,巢式PCR的引入减少了杂带量。结论获得构建金属还原酶基因缺失所需的融合片段,为实现烟曲霉金属还原酶基因的敲除与功能研究奠定基础。巢式-重叠PCR可以提高产物的特异性。

Objective To construct DNA fusion fragments for metalloreductase gene disruption in Aspergillus fumigatus by SOE PCR, and determine the effects of nested PCR on SOE PCR. Methods The 5＇-region and 3＇-region of metalloreductase gene sequences were analyzed using sequence alignment and regions with high homology identified. Primers were designed in these regions to amplify the selected 5＇-region and 3＇-region of metalloreduetase gene. At the same time, two DNA fragments were syn- thesized by PCR in hygromycin resistance marker cassette. By using corresponding DNA fragments as a template, fusion DNA fragments were amplified by nested PCR. The fusion DNA fragments were analyzed by gel electrophoresis and sequencing. Re- sults The sizes of fusion DNA fragments were correct according to gel eleetrophoresis results. And it was verified that the fusion DNA fragments were linked correctly and were in high similarity with designed sequences by sequencing analysis. In contrast with general SOE PCR, the association of nested PCR reduced the amount of incorrect sized bands. Conclusion The DNA fusion fragments for metalloreductase gene disruption in Aspergillus fumigatus are obtained, which provide the foundation for disruption and investigation of metalloreduetase gene function. Nested SON PCR can increase the specificity of PCR products.