Division of Gastroenterology, Union Hospital of Fujian Medical University, Fujian Province, China.
Mol Biol Rep. 2010 Mar;37(3):1421-5. doi: 10.1007/s11033-009-9527-1. Epub 2009 Mar 31.
In the post-genomic era, providing a detailed description of protein functions poses a formidable challenge. To gain functional insights, we have to construct many kinds of expression vectors. DNA recombination based on polymerase chain reaction (PCR) and digestion followed by ligation is the preferred method for vector construction. However, this existing pattern is intrinsically limited by the selection of restriction endonuclease, prompting researchers to use commercial suppliers rather than amplifying genes themselves. Moreover, this method would introduce additional bases into the PCR products, which may be undesired for the construction of epitope expressing plasmid. A PCR-based gene synthesis method, referred to as competitive priming PCR (CP-PCR), is described here to efficiently assemble the plasmid expressing fusion protein of fibrinogen alpha chain and binding domain of galactose transcription factor. A pair of competitive sense primers were designed for the same target sequence. With the presence of antisense primer, PCR amplification of target sequence was performed in the same one system. The PCR product was underwent single digestion by using PstI, followed by ligation with the vector pCMV-BD linearized with EcoRIand PstI. The reconstructed plasmid was validated by sequencing and the fusion protein was affirmed by western blot. CP-PCR combines the superior convenience and precision of PCR. Moreover, it is perfectly capable of generating nearly all kinds of cohesive terminuses, which are ready to recombination in the presence of single digestion or even in the absence of digestion. We demonstrate, by using CP-PCR, the feasibility of directed cloning interested sequence only in the requirement of single digestion or even in the absence of digestion. Competitive priming PCR is demonstrated with convenience and precision equivalent to the traditional method. More than that, "seamless" DNA recombination may be achieved by this novel strategy.
在后基因组时代，详细描述蛋白质功能是一个艰巨的挑战。为了获得功能见解，我们必须构建许多种表达载体。基于聚合酶链反应 (PCR) 和酶切后连接的 DNA 重组是构建载体的首选方法。然而，这种现有模式本质上受到限制内切酶选择的限制，促使研究人员使用商业供应商而不是扩增自己的基因。此外，这种方法会在 PCR 产物中引入额外的碱基，这对于构建表位表达质粒可能是不希望的。这里描述了一种基于 PCR 的基因合成方法，称为竞争引物 PCR (CP-PCR)，用于有效地组装表达纤维蛋白原α链融合蛋白和半乳糖转录因子结合域的质粒。针对同一目标序列设计了一对竞争 sense 引物。在反义引物存在的情况下，在同一个系统中进行目标序列的 PCR 扩增。使用 PstI 对 PCR 产物进行单酶切，然后用 EcoRI 和 PstI 线性化的载体 pCMV-BD 进行连接。通过测序验证了重建质粒，并用 Western blot 验证了融合蛋白。CP-PCR 结合了 PCR 的卓越便利性和精确性。此外，它完全能够产生几乎所有类型的粘性末端，这些末端在单酶切甚至无需酶切的情况下都可以进行重组。我们通过使用 CP-PCR 证明了仅在单酶切甚至无需酶切的要求下定向克隆感兴趣序列的可行性。CP-PCR 具有与传统方法相当的便利性和精确性。不仅如此，这种新策略还可以实现“无缝”DNA 重组。
