吉布森装配插入法提高了长DNA和多片段重叠延伸PCR的扩增效率。

IF 2.2 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS BioTechniques Pub Date : 2023-06-01 DOI:10.2144/btn-2023-0012
Junyi Liu, Fangyin Liu, Xueer Luo, Ming Chen, Chengjun Wang, Liuyue Wang, Huabo Chen
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引用次数: 0

摘要

对于困难的重叠延伸PCR,在两轮PCR之间插入一个Gibson组装过程,以促进在中等温度下形成完整的基因模板。也就是说,在扩增每个DNA片段后,它们按相同比例通过吉布森组装过程被排除。然后,将组装好的混合物用作第二轮PCR的模板。以视网膜母细胞瘤基因的单位点和双位点突变克隆为例,验证了这一观点。该方案将重叠延伸PCR与Gibson组装巧妙地结合在一起,显著提高了基因扩增效率,特别是在使用重叠延伸PCR进行长基因和多片段融合时。
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Gibson assembly interposition improves amplification efficiency of long DNA and multifragment overlap extension PCR.

For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

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来源期刊
BioTechniques
BioTechniques 工程技术-生化研究方法
CiteScore
4.40
自引率
0.00%
发文量
68
审稿时长
3.3 months
期刊介绍: BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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