Fast and precise multi-site mutagenesis on linear DNA fragments
The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant stran...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2024-12-01
|
| Series: | Biotechnology & Biotechnological Equipment |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/13102818.2024.2385423 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850246033088446464 |
|---|---|
| author | Jie Zhang Youhui Yang Zhaoguan Wang Qian Liu Peixian Li Shaohua Kang Hao Qi |
| author_facet | Jie Zhang Youhui Yang Zhaoguan Wang Qian Liu Peixian Li Shaohua Kang Hao Qi |
| author_sort | Jie Zhang |
| collection | DOAJ |
| description | The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library. |
| format | Article |
| id | doaj-art-fbaf98adaeb846c28db79cd49962d443 |
| institution | OA Journals |
| issn | 1310-2818 1314-3530 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Biotechnology & Biotechnological Equipment |
| spelling | doaj-art-fbaf98adaeb846c28db79cd49962d4432025-08-20T01:59:17ZengTaylor & Francis GroupBiotechnology & Biotechnological Equipment1310-28181314-35302024-12-0138110.1080/13102818.2024.2385423Fast and precise multi-site mutagenesis on linear DNA fragmentsJie Zhang0Youhui Yang1Zhaoguan Wang2Qian Liu3Peixian Li4Shaohua Kang5Hao Qi6Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaDepartment of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR ChinaThe effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library.https://www.tandfonline.com/doi/10.1080/13102818.2024.2385423Multisite mutagenesisalkaline denaturationsingle-stranded DNAGFP |
| spellingShingle | Jie Zhang Youhui Yang Zhaoguan Wang Qian Liu Peixian Li Shaohua Kang Hao Qi Fast and precise multi-site mutagenesis on linear DNA fragments Biotechnology & Biotechnological Equipment Multisite mutagenesis alkaline denaturation single-stranded DNA GFP |
| title | Fast and precise multi-site mutagenesis on linear DNA fragments |
| title_full | Fast and precise multi-site mutagenesis on linear DNA fragments |
| title_fullStr | Fast and precise multi-site mutagenesis on linear DNA fragments |
| title_full_unstemmed | Fast and precise multi-site mutagenesis on linear DNA fragments |
| title_short | Fast and precise multi-site mutagenesis on linear DNA fragments |
| title_sort | fast and precise multi site mutagenesis on linear dna fragments |
| topic | Multisite mutagenesis alkaline denaturation single-stranded DNA GFP |
| url | https://www.tandfonline.com/doi/10.1080/13102818.2024.2385423 |
| work_keys_str_mv | AT jiezhang fastandprecisemultisitemutagenesisonlineardnafragments AT youhuiyang fastandprecisemultisitemutagenesisonlineardnafragments AT zhaoguanwang fastandprecisemultisitemutagenesisonlineardnafragments AT qianliu fastandprecisemultisitemutagenesisonlineardnafragments AT peixianli fastandprecisemultisitemutagenesisonlineardnafragments AT shaohuakang fastandprecisemultisitemutagenesisonlineardnafragments AT haoqi fastandprecisemultisitemutagenesisonlineardnafragments |