TY - JOUR
T1 - Site-directed mutagenesis
AU - Nøhr, Jane
AU - Kristiansen, Karsten
N1 - Keywords: DNA; Escherichia coli; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Templates, Genetic
PY - 2003
Y1 - 2003
N2 - Site-directed mutagenesis (SDM) is a powerful tool for analyzing protein structure and function, protein folding, and enzyme mechanism (1). Several protocols for SDM by using polymerase chain reaction (PCR) are available (2-4), resulting in protocols that are faster, more efficient, and less tedious. The major problem encountered in site-directed mutagenesis is, however, not the introduction of the mutations but the separation of the mutagenized DNA from the wild-type DNA that served as a template. One of the fastest and most powerful mutagenesis and selection strategies is QuikChangeTM developed by Stratagene Inc. (La Jolla, CA). The protocol shown in this chapter was developed from this method and allows rapid introduction of mutations (insertions, deletions, and exchanges) into a sequence of interest, using one or two complementary mutagenesis primers to amplify the entire plasmid in a single PCR reaction. This is followed by the destruction of the parental wild-type template by DpnI digestion and the direct transformation into Escherichia coli cells. This strategy allows introduction and selection of desired mutants in as little as 24 h.
AB - Site-directed mutagenesis (SDM) is a powerful tool for analyzing protein structure and function, protein folding, and enzyme mechanism (1). Several protocols for SDM by using polymerase chain reaction (PCR) are available (2-4), resulting in protocols that are faster, more efficient, and less tedious. The major problem encountered in site-directed mutagenesis is, however, not the introduction of the mutations but the separation of the mutagenized DNA from the wild-type DNA that served as a template. One of the fastest and most powerful mutagenesis and selection strategies is QuikChangeTM developed by Stratagene Inc. (La Jolla, CA). The protocol shown in this chapter was developed from this method and allows rapid introduction of mutations (insertions, deletions, and exchanges) into a sequence of interest, using one or two complementary mutagenesis primers to amplify the entire plasmid in a single PCR reaction. This is followed by the destruction of the parental wild-type template by DpnI digestion and the direct transformation into Escherichia coli cells. This strategy allows introduction and selection of desired mutants in as little as 24 h.
U2 - 10.1385/1-59259-394-1:127
DO - 10.1385/1-59259-394-1:127
M3 - Journal article
C2 - 12840545
SN - 1064-3745
VL - 232
SP - 127
EP - 131
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -