chmi 4226e - w20051 recombinant dna technology chmi 4226 e week of march 2, 2009 mutagenesis
TRANSCRIPT
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 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.