CHMI 4226E - W20051 Recombinant DNA Technology CHMI 4226 E Week of March 2, 2009 Mutagenesis

CHMI 4226E - W20052 cDNA clones! Ori C Amp R Sequencing Restriction mapping Sub-cloning Identification Expressed sequence tag (EST) mRNA expression Mutagenesis Complete? Blast probe 5’/3’ RACE

CHMI 4226E - W20053 Site-directed Mutagenesis

CHMI 4226E - W20054 Use of ung - E. coli Strains

CHMI 4226E - W20055 PCR-mediated mutagenesis Very useful to introduce insertions and deletions, as well as point mutations. Makes use of modified primers which bear the desired point mutation OR insertion/deletion.

CHMI 4226E - W20056 Error-prone PCR Modified form a PCR in which conditions are altered to decrease the fidelity of the polymerase: –Non-proofreading polymerase (Taq DNA Pol) –Low annealing temperature –Low/unequel concentrations of dNTPs –High Mg +2 –High cycle number Allows for the creation of a library of mutated PCR products, which are cloned in the vector of interest; Screening is then performed to search for PCR products with alterations in the desired properties.