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A Positive/Negative Selection Cassette for Red Recombination of BAC Clones.
- Source :
-
Applied Biochemistry & Microbiology . Aug2024, Vol. 60 Issue 4, p757-764. 8p. - Publication Year :
- 2024
-
Abstract
- Conventional gene cloning methods always require tedious processes confined by PCR conditions and restriction sites, which might result in a large number of redundant fragments in the constructs. These shortages can be overcome by using DNA recombination methods. Homologous recombination in Escherichia coli cells was used to develop positive/negative cassette for phage λ red operon recombination. In the bi-functional cassette, the Ala294Gly α-subunit mutant of phenylalanyl-tRNA synthetase (mPheS) was fused with the kanamycin-resistant (KanR) cassette under the control of the promoter of the E. coli gene NEOKAN as a single open reading frame (ORF) by PCR. KanR was utilized as a positive selection marker, while the mutant gene mPheS served as a negative selection marker. Two-step red-mediated recombination was used to replace the third exon of TMEM18 in the BAC clone RP23-25C13 with eGFP. Initially, the third TMEM18 exon in the BAC was replaced with the NEOKAN-mPheS-KanR cassette, and 40% of the clones on the positive selection plate (LB plate with kanamycin) were positive. The second step involved replacing the NEOKAN-mPheS-KanR cassette with eGFP in the modified BAC, and 60% of the clones on the negative plate (p-Cl-phe plate) were positive. Following the two steps, eGFP was used to replace the third exon of TMEM18 in the BAC. The NEOKAN-mPheS-KanR could be a promising choice as a positive/negative selection cassette for red recombination to establish constructs and make gene manipulation on BAC more applicable and effortless. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00036838
- Volume :
- 60
- Issue :
- 4
- Database :
- Academic Search Index
- Journal :
- Applied Biochemistry & Microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 178657227
- Full Text :
- https://doi.org/10.1134/S0003683823602846