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Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

ABSTRACT: PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair 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?. 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 5' 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 biology-oriented laboratories.

SUBMITTER: Jacobus AP

PROVIDER: S-EPMC4361335 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

Jacobus Ana Paula AP Gross Jeferson J

PloS one 20150316 3

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair 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 DH5α. We found th ...[more]

PMID: 25774528

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Project description:BACKGROUND:Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. RESULTS:We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. CONCLUSIONS:Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional evidence to explain the relationship between genomic variation and ecological adaptation.

Dataset Information

Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

Publications

Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

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