A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments
Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into Escherichia coli. This...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2022-11-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/btn-2022-0085 |
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| _version_ | 1850154049414889472 |
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| author | Zhibo Yang Zan Chen Yueping Zhang |
| author_facet | Zhibo Yang Zan Chen Yueping Zhang |
| author_sort | Zhibo Yang |
| collection | DOAJ |
| description | Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into Escherichia coli. This method successfully generated mutations for plasmids of 8.3 kb and 11.0 kb with high efficiencies. The method only requires Q5 DNA polymerase and DpnI, which greatly reduces costs. The procedure is simple, including PCR reaction, DpnI treatment and transformation. This simple, efficient and economical site-directed mutagenesis method for large plasmids is likely to be widely applied in the future. |
| format | Article |
| id | doaj-art-7527ab267eb84f61ba1b7626269fd1e1 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2022-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-7527ab267eb84f61ba1b7626269fd1e12025-08-20T02:25:34ZengTaylor & Francis GroupBioTechniques0736-62051940-98182022-11-0173523924510.2144/btn-2022-0085A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragmentsZhibo Yang0Zan Chen1Yueping Zhang21College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China1College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China1College of Veterinary Medicine, China Agricultural University, Beijing, 100193, ChinaDespite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into Escherichia coli. This method successfully generated mutations for plasmids of 8.3 kb and 11.0 kb with high efficiencies. The method only requires Q5 DNA polymerase and DpnI, which greatly reduces costs. The procedure is simple, including PCR reaction, DpnI treatment and transformation. This simple, efficient and economical site-directed mutagenesis method for large plasmids is likely to be widely applied in the future.https://www.future-science.com/doi/10.2144/btn-2022-0085Escherichia coliGibson assemblyin vivo assemblyin vivo recombinationoverlapping extension PCRPCR-based site-directed mutagenesis |
| spellingShingle | Zhibo Yang Zan Chen Yueping Zhang A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments BioTechniques Escherichia coli Gibson assembly in vivo assembly in vivo recombination overlapping extension PCR PCR-based site-directed mutagenesis |
| title | A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments |
| title_full | A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments |
| title_fullStr | A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments |
| title_full_unstemmed | A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments |
| title_short | A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments |
| title_sort | simple and economical site directed mutagenesis method for large plasmids by direct transformation of two overlapping pcr fragments |
| topic | Escherichia coli Gibson assembly in vivo assembly in vivo recombination overlapping extension PCR PCR-based site-directed mutagenesis |
| url | https://www.future-science.com/doi/10.2144/btn-2022-0085 |
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