摘要
融合PCR技术(fusion PCR)采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以3个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行3个片段及4个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。
Fusion PCR, which employs chimeric primers to generate PCR products with complementary ends in its amplifications, is a rapid and flexible method in joining different DNA fragments. This method can assemble DNA fragments without the treatment of restriction endonucleases and T4 DNA ligase. It offered a shortcut for the construction of homologous recombinant fragments. Through assembling three recombinant fragments successfully, fusion PCR procedure was improved and manipulation essentials of fusion PCR were described in detail. The results indicated that the improved procedure can assemble three or four fragments simultaneously, and the length of each fusion product is above 4.5 kb. The result recombinants were be use in further experiments, which structures had been confirmed by sequencing.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第8期53-58,共6页
China Biotechnology
关键词
融合PCR
重组片段
同源臂
抗性基因
Fusion PCR Recombinant fragment Homologous arm Resistant gene
