A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

Abstract Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP met...

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Autores principales:	Kewei Zhang, Xiaomei Yin, Kaituo Shi, Shihua Zhang, Juan Wang, Shasha Zhao, Huan Deng, Cheng Zhang, Zihui Wu, Yuan Li, Xiangyu Zhou, Wensheng Deng
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/56bcfbaa0c08433d9b16cd995444f35d
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spelling	oai:doaj.org-article:56bcfbaa0c08433d9b16cd995444f35d2021-12-02T15:45:21ZA high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation10.1038/s41598-021-89884-z2045-2322https://doaj.org/article/56bcfbaa0c08433d9b16cd995444f35d2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89884-zhttps://doaj.org/toc/2045-2322Abstract Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.Kewei ZhangXiaomei YinKaituo ShiShihua ZhangJuan WangShasha ZhaoHuan DengCheng ZhangZihui WuYuan LiXiangyu ZhouWensheng DengNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kewei Zhang Xiaomei Yin Kaituo Shi Shihua Zhang Juan Wang Shasha Zhao Huan Deng Cheng Zhang Zihui Wu Yuan Li Xiangyu Zhou Wensheng Deng A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
description	Abstract Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.
format	article
author	Kewei Zhang Xiaomei Yin Kaituo Shi Shihua Zhang Juan Wang Shasha Zhao Huan Deng Cheng Zhang Zihui Wu Yuan Li Xiangyu Zhou Wensheng Deng
author_facet	Kewei Zhang Xiaomei Yin Kaituo Shi Shihua Zhang Juan Wang Shasha Zhao Huan Deng Cheng Zhang Zihui Wu Yuan Li Xiangyu Zhou Wensheng Deng
author_sort	Kewei Zhang
title	A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
title_short	A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
title_full	A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
title_fullStr	A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
title_full_unstemmed	A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation
title_sort	high-efficiency method for site-directed mutagenesis of large plasmids based on large dna fragment amplification and recombinational ligation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/56bcfbaa0c08433d9b16cd995444f35d
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A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

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