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

VL - 232

SP - 127

EP - 131

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -