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 DH5α. 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.

Optimization of the experimental parameters of the ligase cycling reaction

“…In contrast to de Kok et al (8), the concentration of the vector pJET1.2/blunt (part 7) was reduced to 0.3 nM to achieve fewer religations of the vector. Positive effects of increasing the molar insert-to-vector ratio were already described for other cloning methods (28,29) and were confirmed in preliminary LCRs (data not shown). LCRs using these experimental concentrations, i.e.…”

Phase I clinical trial of idiotypic DNA vaccine administered as a complex with polyethylenimine to patients with B-cell lymphoma

“…Alternatively, the scFv fragment is cloned directly into the vector by OE-PCR and homology recombination in E.coli. 29 The assembled plasmids are verified by restriction mapping and DNA sequencing. Extraction of plasmids is performed by MaxiPrep Kit (Life Technologies).…”

<i>In vivo</i> cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in <i>Salmonella enterica</i>

“…There are several examples (Bubeck et al, 1993;Jacobus and Gross, 2015;Li et al, 2011;Oliner et al, 1993;Spiliotis, 2012;Thieme et al, 2011) of recombineering plasmids, including BAC (Narayanan and Chen, 2011). However, the technique described here is unique because P22 transduction and Flp-mediated site-specific recombination are the keys and because large chromosomal segments can be directly cloned into a derivative of BAC without isolating, amplifying, or digesting the target chromosome.…”

“…enzymes, which vary from insert to insert even for a given vector plasmid, and there is no need to purify and ligate DNA fragments, which is required in conventional recombinant techniques. This method, which relies on PCR amplification for the preparation of entire insert fragments (Bubeck et al, 1993;Jacobus and Gross, 2015;Li et al, 2011;Oliner et al, 1993;Spiliotis, 2012;Thieme et al, 2011) or entire plasmid clones (Jacobus and Gross, 2015;Li et al, 2011;Spiliotis, 2012), co-transformation, and recombinases, such as RecE/RecT from Rac prophage (Zhang et al, 1998) or Redabd from bacteriophage lambda (Bubeck et al, 1993;Jacobus and Gross, 2015;Li et al, 2011;Oliner et al, 1993;Spiliotis, 2012;Thieme et al, 2011), also allows several fragments to be combined into a vector at a time, thus reducing the cloning processing time. However, due to the high dependence on PCR, which produces errors in vitro and tends to amplify smaller fragments at a higher yield, its use for DNA fragments larger than 10 kb or so is unreliable.…”

“…Then, to facilitate double crossover events at the target gene on the chromosome, this linear DNA fragment is transformed into bacterial cells expressing lambda Red recombinase. Because the cassette was designed to harbor two FRT sites at the both ends of the antibiotic marker, it can be removed In vivo cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in Salmonella enterica (Received March 5, 2016;Accepted April 12, 2016; J-STAGE Advance publication date: September 23, 2016) Akinori Kato* Agriculture,Kindai University,Nara,Japan Introduction Recombineering-based in vivo specific cloning methods (Bubeck et al, 1993;Jacobus and Gross, 2015;Li et al, 2011;Oliner et al, 1993;Spiliotis, 2012;Thieme et al, 2011) are capable of difficult constructions because it is unnecessary to select possible combinations of restriction from the target gene disruption site by Flp recombinase expressed from the pCP20 plasmid (Cherepanov and Wackernagel, 1995), leaving a single FRT, "scar" (Datsenko and Wanner, 2000). Thus, the cassette used for the first run can be reused for following gene disruption events by additional one-step inactivation to generate multiple gene deletion mutations on different chromosomal locations of a strain.…”