DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

Abstract

Site-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombinationmediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR.Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

DNA gap repair in Escherichia coli for multiplex site‐directed mutagenesis

“…As shown in Figure 4, DNA denaturation significantly decreased colony counts in both recombineering‐ and recombineering+ cells when the gap was repaired with a 10‐nt hArm ssODN, but there was no difference in 20 and 30‐nt hArm ssODNs. Based on these results, we conclude that denatured DNA is less active in DGR but increasing hArm size helps to restore the activity, possibly by facilitating the renaturation of vector DNA 18 …”

“…In DeGeRing, targeted DNA is linear. This DNA cannot be maintained intracellularly because E coli lacks both non‐homological end joining and the ability to replicate linear DNA 18 . However, it is well known that linear plasmid DNA can be established in E coli cells by intramolecular DNA recircularization with accompanying microdeletions 30,31 .…”

Live‐cell PCR and one‐step purification streamline DNA engineering

“…For example, among cells coelectroporated with gap containing plasmid DNA and 21-nt targeting oligonucleotide, only approximately every 7th cell was correctly repaired, while others contained background plasmids with short microdeletions. 10 These background plasmids could not be detected by gel electrophoresis of 8.5 kb DNA of either native or linearized plasmids (Figure 2A,B). This inability is not either extraction or bacterial strain-specific.…”

“…6 The use of PCR for the introduction of a gap into wild-type Tnnt2 PAC construct and DGR with ssODNs has been described previously. 10 Linear PCR derived DNA was electroporated at 12 000-13 000 V/cm at a time constant of 10 ms, covalently closed circular DNA was electroporated at 17 000-18 000 V/cm at a time constant of 7.5 ms using BioRad Gene Pulser Xcell into 10 μL of electrocompetent cells. Targeted PCR derived DNA before electroporation was concentrated and purified by silica columns or one-step LiCl-isopropanol DNA precipitation as described below and washed with 70% EtOH.…”

“…Background plasmids are typically characterized by additional microdeletions. 10 To the best of our knowledge, there is currently no reliable end-point PCR assay to screen for plasmids containing the desired point mutations, but PCR products containing microdeletions can be detected, thereby allowing the elimination of background plasmids prior to DNA sequencing (Figure 1).…”