Linker-scanning mutagenesis of DNA.
Two protocols are described in which clusters of point mutations are introduced throughout a sequence of interest that has been cloned into a plasmid vector. The first protocol uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides are synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence actually provides the desired clusters of point mutations as it is moved or "scanned" across the region by its position at the varied endpoints of the deletion mutation series. An Alternate Protocol makes use of site-directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the target region.
Greene
J M
JM
Massachusetts General Hospital, Boston, Massachusetts, USA.
D005821:Genetic Techniques; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed