Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes

2020

FASEB j.

Self Cite

In vivo DNA engineering such as recombineering (recombination‐mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR‐mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one‐step LiCl‐isopropanol purification can streamline DNA engineering. In DNA gap repair, live‐cell PCR allowed the convenient elimination of clones containing background plasmids prior to DNA sequencing. Live‐cell PCR also enabled the generation of specific DNA sequences for DNA engineering up to 11 kilo base pairs in length and with up to 80 base pair terminal non‐homology. Using gel electrophoresis and DNA melting curve analysis, we showed that LiCl‐isopropanol DNA precipitation removed primers and small, nonspecific PCR products from live‐cell PCR products in only ~10‐minutes. DNA sequencing of purified products yielded Phred quality scores values of ~55%. These data indicate that live‐cell PCR and LiCl‐isopropanol DNA precipitation are ideal to prepare DNA for sequencing and other downstream DNA applications, and might therefore accelerate high‐throughput DNA engineering pipelines.

Section: Discussionmentioning

confidence: 99%

Section: Dna Engineeringmentioning

confidence: 99%

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

2020

FASEB j.

Self Cite

“…Electro-competent cells were prepared as described previously 6 . DNA was electroporated at 17,000-18,000 V/cm at time constant 7.5 msec using BioRad Gene Pulser Xcell into 10 μl competent cells.…”

Section: Bacterial Transformationmentioning

confidence: 99%

“…After denaturing DNA was precipitated with EtOH and washed with 70% EtOH, the DNA pellet was dissolved in electroporation buffer 6…”

Section: Bacterial Transformationmentioning

confidence: 99%

See 1 more Smart Citation

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

2018

Preprint

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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

2020

FASEB j.

Self Cite

Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including nonbiased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10 -6 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics. K E Y W O R D Sgap repair cloning, in vivo DNA engineering, protein engineering, reverse genetics 6352 | LYOZIN aNd BRUNELLI How to cite this article: Lyozin GT, Brunelli L. DNA gap repair in Escherichia coli for multiplex site-directed mutagenesis. The FASEB Journal. 2020;34:6351-6368.