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...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2024-12-01
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| Series: | Biotechnology & Biotechnological Equipment |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/13102818.2024.2385423 |
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| Summary: | 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. |
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| ISSN: | 1310-2818 1314-3530 |