Date Published: January 23, 2019
Publisher: Public Library of Science
Author(s): Ruhu Qi, Gottfried Otting, Ruslan Kalendar.
The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3’-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.
Faithful linearization and amplification of whole plasmids by PCR with high-fidelity DNA polymerases present the basis of modern cloning and mutagenesis methods such as sequence and ligation independent cloning (SLIC) [1,2], Gibson assembly , and QuikChange (Agilent, La Jolla, CA) . Optimized kits have become available commercially, but often the details of the optimizations have not been described. Having established an in-house protocol for SLIC to save costs for large numbers of reactions, we noticed that the cloning success rate was highly variable and dependent on the batch of T4 DNA polymerase used, indicating that the protocol could be upset by small changes in the 3’-exonuclease activity of the enzyme, which SLIC relies on. This suggested that better control of the enzyme’s activity would render the SLIC protocol more robust. In different published versions of the protocol, the activity of the T4 DNA polymerase was adjusted by adjusting the temperature and duration of treatment [1,2,5–7]. T4 DNA polymerase is a highly active exonuclease, which has been shown to completely digest primers of 17 nucleotide length in 15 seconds at 37 °C in experiments, where a two-fold excess of the enzyme was presented with a short primer/template construct . Such high digestion rates make it difficult to limit the digestion to about 20 nucleotides. In the present work we show that (i) using the wild-type enzyme at a concentration lower than that of the DNA leads to highly non-uniform DNA digestion and (ii) an attenuated mutant offers better control of the exonuclease activity while delivering single-stranded DNA overhangs of uniform length, which is beneficial for cloning with the SLIC method as well as for mutagenesis using QuikChange.
The present work introduces the mutant Y320A of T4 DNA polymerase (E2 enzyme) for cloning and site-directed mutagenesis. Owing to its slower 3’-exonuclease rate, the E2 enzyme can be used in large excess relative to the DNA to generate uniform single-stranded DNA overhangs without losing control over the digestion rate, which is important for, e.g., manual execution of multiple cloning experiments in parallel. In our hands and compared with conventional restriction–ligation methods, the RQ-SLIC protocol is at least as reliable while providing significant savings in time and cost. Incubation with the E2 enzyme and subsequent heat inactivation can conveniently be performed on a PCR thermocycler.
SLIC and QuikChange methods are convenient for their independence from restriction sites, by relying on endogenous DNA polymerase and ligase in E. coli to fill in and ligate the modified double-stranded plasmid after transformation. The present work shows that the success of both methods can be enhanced, without relying on proprietory components of costly kits, by using the 3’-exonuclease activity of the Y320A mutant of T4 DNA polymerase to generate single-stranded DNA overhangs. The attenuated activity of this mutant delivers uniform digestion rates of DNA under more convenient conditions of temperature and timing than the wild-type enzyme. The resulting improved control of enzymatic activity enhances the reliability of existing cloning and mutagenesis protocols and translates into corresponding savings in time and expense.