重叠延伸PCR方法的建立与应用

目的建立一种新型PCR技术（重叠延伸PCR）并用于腺病毒早期基因E1A与内核蛋白体进入位点IRES（internal ribosome entry site）的直接连接,为构建复制型腺病毒载体作准备.方法以PcAE1A质粒与PIRES-EGFP质粒为模板,通过引物设计,使E1A基因3＇端与IRES基因5＇端具有相互重叠的一段序列,用该组引物行PCR分别扩增E1A基因与IRES基因,再以这两种PCR产物的混合物作为模板,进行重叠延伸PCR扩增E1A-IRES基因片段.结果经酶切及测序鉴定重叠延伸PCR方法成功构建了E1A-IRES基因片段.结论重叠延伸PCR法能将任意两段DNA序列连接起来,其在分子生物学的领域中具有潜在应用价值.

Establishment and application of overlap-extension PCR

Objective To establish a novel PCR technique (overlap-extension PCR) and link gene E1A with gene IRES (internal ribosome-entry site) for the preparation of construction of replication adenovirus vector by the technique.Methods Using the PcAElA plasmid and PIRES-EGFP plasmid as the template, the gene E1A and IRES were amplified respectively. As designed,the antisense primer for gene E1A was complementary with sense primer for gene IRES.Therefore, the 3' end of gene E1A had a sequence that was overlapped with the 5' end of gene IRES.The PCR products for gene E1A and IRES were mixed as template, and the sense primer for gene EIA and antisense primer for gene IRES were used together to initiate the synthesis of the gene fragment E1A-IRES. Results As desired, the gene fragment E1A-IRES was obtained by the novel PCR technique.Conclusion The overlap-extension PCR can link any two DNA fragments and the technique has the potential application value in the field of molecular biology.