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
What plasmids do we use for plasmid shuffle?
Life support plasmid has a selectable marker and the WT sequence of YFG
Plasmid of interest has mutant yfg and a different selectable marker
How do you do plasmid shuffle?
- Transform the life support and mutant plasmid into a strain auxotrophic for both selectable markers and knocked out YFG
- Plate the transformants on 5FOA to select for those that lose the life support plasmid
- Assess the functionality of the survivors
What are the 2 key components that are endogenously in cells and are required for RNAi to work?
Dicer and RISC
How does RNAi work?
Double stranded RNA gets processed into short interfering RNA by Dicer, and interacts with the RISC complex and binds to its target mRNA. The target mRNA gets degraded and knocks down gene expression
What are 3 ways to get dsRNA into C. elegans?
- Transform E coli with a vector encoding a sense and antisense strand, then feed the E coli to the worms
- Give the worms a bath in it
- Inject it directly into the worms
What are the disadvantages of RNAi?
Often have off target effects, so need to use multiple dsRNA that bind in multiple places along the target mRNA to avoid that
What is gene tagging?
Sticking something onto either the C term or N term of a protein of interest to track it or use antibodies
Is it easier to tag the C term or the N term of a protein?
C term
What sequences on the C-term gene tagging template will be amplified when you do PCR?
The sequence of the tag and a selectable marker
What primers do you use to create the targeting vector for C-term gene tagging?
The 3’ ends are completely complementary to the template, and the 5’ ends match sequences in and around YFG. The 5’ primer matches sequences in YFG, and the 3’ primer matches sequences downstream of YFG
What happens when you transform your cells with the C-term gene tagging targeting vector?
You get ends out recombination and the tag gets fused to the C-term of YFG, generating a fusion protein
Why is N-term tagging harder to do?
The gene replacement can push the promotor too far upstream to do anything
How can we fix the problem with N-term tagging where the promotor is too far upstream to transcribe the fusion protein?
- Have an inducible promotor on your vector between Ura3 and GFP, which will be right in front of the fusion proteins
- Have a copy of GFP on either side of URA3 and select for rare internal recombination events that result in the deletion of URA3, and the promotor is brought back to the fusion protein
What tag do we add to a protein if we want to make a temperature sensitive protein?
Arg-DHFR. Is stable at 25°C but gets degraded at 37°C
