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

Abstract

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.