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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

Full text
Author(s):
Jacobus, Ana Paula [1] ; Gross, Jeferson [1]
Total Authors: 2
Affiliation:
[1] Univ Sao Paulo, Dept Biol Sci, Luiz de Queiroz Coll Agr, Piracicaba, SP - Brazil
Total Affiliations: 1
Document type: Journal article
Source: PLoS One; v. 10, n. 3 MAR 16 2015.
Web of Science Citations: 31
Abstract

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gaprepair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5 alpha. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 50 termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biologyoriented laboratories. (AU)

FAPESP's process: 13/15743-9 - The evolutionary impact of sexual reproduction across 5,000 generations of yeast and its application to engineer ethanol tolerant strains
Grantee:Jeferson Gross
Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants