Gene to phenotype Flashcards
What is site directed mutagenesis?
Making a specific change to a DNA sequence
Why is site directed mutagenesis useful?
Investigate structure/function relationships, or for engineering fusion proteins
What does site directed mutagenesis require before starting?
Detailed knowledge of the gene sequence
What type of primers do we use for Quik-change site-directed mutagenesis?
Mutagenic primers. Sequences are completely complementary except for where we want to introduce the mutation
Where in the primer do we want to put our desired mutation for Quik-change site-directed mutagenesis?
Somewhere in the middle so we get good annealing
What type of polymerase do we use for Quik-change site-directed mutagenesis? Why?
PfuUltra. Has high fidelity and makes very few mistakes, and is processive and stays on the strand until its done. Also doesn’t displace the 5’ end of the primer
What are the 3 PCR products produced after 16 cycles for Quik-change site-directed mutagenesis?
Parental homoduplexes, heteroduplexes, and mutant homoduplexes
How do we differentiate the 3 PCR products for Quik-change site-directed mutagenesis?
Differential methylation. The original strands were likely cloned in E. coli, so they’ll be methylated. The parental homoduplexes are fully methylated, the heteroduplexes are hemimethylated, and the mutant homoduplexes are not methylated at all. If we use Dpn1, it will digest all methylated or hemi-methylated DNA and leave only the mutant duplexes left
Which restriction enzyme digests methylated DNA?
Dpn1
What do we do with the newly made plasmids with the introduced mutation in Quik-change site-directed mutagenesis?
Transform them into E coli to repair the nicks, then miniprep it out to transform into your organism and look for a phenotype
What are the advantages of Quik-change site-directed mutagenesis?
Uses double stranded plasmids, can use whatever plasmid we want, selects for mutant strain (no screening)
What are the disadvantages of Quik-change site-directed mutagenesis?
Only works if there’s differential methylation so you need an E coli strain that will methylate stuff
How do you do the two step method to introduce a new mutant allele into yeast?
Same idea as targeting YIP integration
- Digest your vector with a enzyme that only cuts once in YFG
- Transform yeast cells
- Plate cells on 5FOA to select for rare recombination events that caused the mutant YFG to end up in the genome and the WT to end up on the plasmid and lost
What is ends out integration?
Sequence is inserted but does not replace what’s already there
What sort of genes do we do plasmid shuffle experiments for?
Essential genes