长距离反向PCR技术高效扩增已知DNA片断的侧翼序列

Long Distance-inverse PCR Technique for Efficient Cloning of Flanking Sequences Adjacent to Known DNA Fragments

为解决传统反向PCR技术扩增片段短、假阳性多的不足,建立了长距离反向PCR(LD I-PCR)扩增技术:0.5μg DNA/mL的反应体系使DNA酶解片段充分自身环化连接,其产物用25 nt^30 nt的序列特异引物进行长距离PCR。结果表明该方法能特异地扩增出长达16 kb的序列,在已知DNA片段的侧翼序列克隆方面具有高效、简便、特异的优点。

To avoid the disadvantages of conventional inverse PCR such as short amplification fragments and pseudopositive products, the long distance-inverse PCR （LD-IPCR） technique was set up as follows. In a 1 mL reaction system, 0.5μg of DNA fragments excised by a single restriction enzyme was adequately self-looped and ligated. Then the reaction products were used as the templates for LD-IPCR amplification using specific inverse primer pairs of 25 - 30 nucleotides. It was proven that this modified technique could successfully make it more efficient, convenient as well as more specific in cloning the flanking sequences adjacent to DNA fragments obtained. The flanking sequence amplified by LD-IPCR could reach 16 kb in length.