CHMI 4226E - W2005 1

Recombinant DNA TechnologyCHMI 4226 E

Week of March 2, 2009

Mutagenesis

CHMI 4226E - W2005 2

cDNA clones!

Ori C

AmpR

Sequencing

Restriction mapping

Sub-cloning

IdentificationExpressed sequence tag

(EST)

mRNA expressionMutagenesis

Complete?

Blast

probe

5’/3’ RACE

CHMI 4226E - W2005 3

Site-directed Mutagenesis

CHMI 4226E - W2005 4

Use of ung- E. coli Strains

CHMI 4226E - W2005 5

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 - W2005 6

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.