Method to Overcome Inefficiencies in Site-Directed Mutagenesis of A/T-Rich DNA. J Biomol Tech 2020 Sep;31(3):94-99
Date
08/25/2020Pubmed ID
32831656Pubmed Central ID
PMC7351327DOI
10.7171/jbt.20-3103-003Scopus ID
2-s2.0-85091559248 (requires institutional sign-in at Scopus site) 2 CitationsAbstract
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.
Author List
DeCero SA 2nd, Winslow CH, Coburn JAuthor
Jenifer Coburn PhD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AT Rich SequenceDNA
Escherichia coli
Mutagenesis, Site-Directed
Oligonucleotides
