• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Molecular Biology Reports >Competitive priming PCR: a versatile method to generate cohesive terminus
【24h】

Competitive priming PCR: a versatile method to generate cohesive terminus

机译：竞争性引物PCR：一种生成内聚末端的通用方法

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

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 α 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. Keywords PCR - Gene cloning
机译：在后基因组时代，提供蛋白质功能的详细描述带来了巨大的挑战。为了获得功能上的见识，我们必须构建多种表达载体。基于聚合酶链反应（PCR）的DNA重组和消化后再连接是载体构建的首选方法。但是，这种现有模式本质上受限制性核酸内切酶选择的限制，促使研究人员使用商业供应商而不是扩增基因本身。而且，该方法会将额外的碱基引入PCR产物中，这对于构建表位表达质粒可能是不希望的。本文描述了一种基于PCR的基因合成方法，称为竞争引物PCR（CP-PCR），可有效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。针对相同的靶序列设计了一对竞争正义引物。在存在反义引物的情况下，在同一系统中进行靶序列的PCR扩增。通过使用PstI对PCR产物进行单次消化，然后与用EcoRI和PstI线性化的载体pCMV-BD连接。通过测序验证重组质粒，并通过western印迹证实融合蛋白。 CP-PCR结合了PCR的卓越便利性和精确性。而且，它完全能够生成几乎所有类型的内聚末端，这些内聚末端在存在单一消化或什至没有消化的情况下即可重组。通过使用CP-PCR，我们证明了仅在单次消化或什至没有消化的情况下定向克隆目的序列的可行性。竞争性引物PCR的简便性和精确性与传统方法相当。除此之外，通过这种新颖的策略可以实现“无缝” DNA重组。关键词PCR-基因克隆

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号