Method to Overcome Inefficiencies in Site-Directed Mutagenesis of A/T-Rich DNA

Abstract

Site-directed mutagenesis (SDM) is an invaluable technique that enables the manipulation of DNA and therefore the primary structure and function of any encoded gene products. Commercial protocols for SDM have been optimized for Escherichia coli and mean A/T content but may hinder generation of desired products using other templates. Mutagenesis of A/T-rich DNA is often hindered by low oligodeoxynucleotide (oligo)-annealing temperatures, requiring oligos longer than manufacturer protocol recommendations. However, longer oligos can result in primer dimer formation and decreased SDM efficiencies. Commercially available kits proved inefficient at generating AT-rich mutants. We sought to generate a modified protocol that generated SDM products detectable using gel electrophoresis and that did not require an apparent limit on oligo length.

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum

“…Amino acid insertions in the Kesv channel were generated by site‐directed mutagenesis as described by DeCero and coworkers 43 using Q5® High‐Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA). PCR products were directly used for the transformation of competent Escherichia coli XL‐1 blue cells by heat shock.…”