Multiplex site-directed mutagenesis strategy including high-efficiency selection of the mutant PCR products.
Site-directed mutagenesis is of great importance for probing the structure/function relationship of proteins. Developing our previous method (Nagy et al. Anal Biochem 324:301-303, 2004), here we report a multiplex strategy for site-directed mutagenesis using PCR in one tube to introduce a single mutation into three or more genes at the same time. DNA fragments carrying the desired mutation can be distinguished from each other in a standard antibiotic selection step of the transformed bacteria. Due to this strategy the mutagenesis procedure for several genes can be accelerated.
Varga-Orvos
Zoltán
Z
Laboratory of Functional Genomics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, Szeged, 6701, Hungary.
Nagy
Zsolt B
ZB
Mészáros
Attila
A
Kökény
Szabolcs
S
Gergely
Péter
P
Tamás
László
L
Poór
Gyula
G
D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012641:Selection, Genetic
D004247:DNA; D004262:DNA Restriction Enzymes