ArticleApplied microbiology and biotechnology2018
Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA).
Article in Applied microbiology and biotechnology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- Cancer and Radiosensitivity Syndromes: Is Impaired Nuclear ATM Kinase Activity the Primum Movens?Cancers · 2022Review
- Alternative Seamless Cloning Strategies in Fusing Gene Fragments Based on Overlap-PCR.Molecular biotechnology · 2021Article
- Cloning Method for Stress-Resistant Gene ofJournal of healthcare engineering · 2021Article
Corrections and comments
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Authors and funding
8 authors.
Funding
Abstract
The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25 bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19 bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.
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