Patch cloning method for multiple site-directed and saturation mutagenesis.

ABSTRACT:

Background

Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.

Results

To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed t

SUBMITTER: Taniguchi N 

PROVIDER: S-EPMC3829206 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature