Site-directed mutagenesis Flashcards
What is site-directed mutagenesis?
intentional mutation of a specific site within a DNA sequence
What are used of SDM when making changes to non-protein coding DNA?
mutate out inconvenient restoration site in middle of gene of interest
investigate importance of specific nucleotide
What are used of SDM when making changes to protein coding DNA?
understand contribution of particular amino acid residue to 3D structure of a protein or its functionality
What info do you need before you can carry out site-directed mutagenesis of protein coding DNA?
DNA sequence of the protein coding gene
3D structure of protein
active site information
activity assay to evaluate mutant enzymes in comparison to wild-type
What are IDT gBlocks?
linear dsDNA molecule
used for Gibson assembly or as PCR template for conventional restriction enzyme cloning
Designing a gBlock that harbours a point mutation?
change codon
copy and paste into an order form & purchase
get gene back and clone into an expression plasmid
What is QuikChange mutagenesis?
clone gene of interest into plasmid
design complementary primers harbouring mutation
PCR = nicked circular strands
treat with DPnI to destroy methylated template plasmid
transform E.coli
Screen transformants
verify mutation
What are the rules for QuikChange primer design?
1) complementary
2) 25-45nt long
3) mutation positioned centrally
4) Tm close to 78c
5) 3’ nucleotide should be a G/C
What are the final stages of QuikChange mutagenesis?
restriction selection
DpnI
used digested mix to transform E.coli
inoculate overnight
What are the steps for EIPCR
1 - clone gene of interest into plasmid
2 - design mutagenic primer with restriction enzyme sites at 5’ ends
3 - perform PCR reaction with primers & plasmid using high-fidelity polymerase
4 - digest
5 - ligate & transform
6 - screen transformants
Rules for EIPCR primer design
anneal back-to-back
15-40nt long
at least 10-15nt of primer should anneal to template
only 1 primer has mutation
What is Alanine scanning?
use site-directed mutagenesis to alter wild-type codon to one specifying alanine to determine contribution of original residue to function of protein
Why is alanine the amino acid used in scanning?
Because the ‘R group’ is only a methyl
What is site-saturation mutagenesis
mutagenesis of wild-type codon to codons specifying the remaining amino acids
What is needed to undertake site-saturation mutagenesis?
- QuikChange or EIPCR
- design 19 sets of primers
- perform reactions
