We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA).
- Authors
Luo, Zhixin; Wang, Shanhe; Jiao, Beilei; Yuan, Dan; Dai, Dongmei; Wang, Lixian; Xu, Kun; Wang, Xin
- 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.
- Subjects
MOLECULAR cloning; GENETIC engineering; MUTAGENESIS; EXONS (Genetics); SIMULATED annealing
- Publication
Applied Microbiology & Biotechnology, 2018, Vol 102, Issue 23, p10119
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-018-9348-z