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Molecular Biology Research Communications، جلد ۱۱، شماره ۱، صفحات ۲۱-۲۷
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عنوان انگلیسی Recombinase-free cloning (RFC) protocol for gene swapping
چکیده انگلیسی مقاله Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC). This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in E. coli host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories.
کلیدواژه‌های انگلیسی مقاله Cloning, Recombinant DNA, Molecular biology, E.coli DH5α
نویسندگان مقاله Hai-Vy Vo-Nguyen |
Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM

Thanh-Tan Nguyen |
Department Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam

Quoc-Gia Mai |
Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM

Thien-Thien Tran |
Department Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam

Thuoc Linh Tran |
Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM

Hieu Tran-Van |
Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM

نشانی اینترنتی https://mbrc.shirazu.ac.ir/article_6552_d80e83e5be7c407e541979c39125a15c.pdf
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