A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

Abstract

BackgroundSite-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.ResultsThe strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.ConclusionThis strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.

Conformational Dynamics‐Guided Loop Engineering of an Alcohol Dehydrogenase: Capture, Turnover and Enantioselective Transformation of Difficult‐to‐Reduce Ketones

“…Site‐Directed Mutagenesis . Site‐directed Mutagenesis of TbSADH was constructed using megaprimer approach with PrimeSTAR DNA polymerase. The PCR conditions for short fragment: 95 °C for 5 min, (95 °C for 30 s, 56 °C for 30 s, 72 °C for 40 s,) ×32 cycles, 72 °C for 10 min.…”

Isoniazid resistance without a loss of fitness in Mycobacterium tuberculosis

“…Two picomoles of E. coli RNA core polymerase (Epicentre) were incubated with 20 pmol of purified sigma factor protein for 30 min in transcription assay buffer (10mM Tris, 50mM KCl, 10mM MgCl 2 , 0.1mM EDTA, 0.25μg/ul BSA), then 0.09 μg of template DNA was added and incubated with 0.35mM 11-biotin UTP, 1mM ATP, 1mM CTP, 1mM GTP and 0.65mM UTP mixture 25 . The template with the putative SigI-binding motif deleted was generated using the mega-primer method 27 .…”

Design of Heteronuclear Metalloenzymes

“…Single mutation site-directed mutagenesis is typically performed using the QuikChange Site Directed Mutagenesis Kit (Sigman et al, 1999). Where applicable, Gibson assembly (Gibson, 2011) and Mega-Primer PCR (Tyagi et al, 2004) can be also used. Detailed descriptions of these methods are beyond the scope of this chapter, and have been covered previously (Séraphin&Kandels-Lewis, 1996, Weissensteiner et al, 2003, Nakayama & Shimamoto, 2014, Casini et al, 2015).…”