Exam stuff! (turn in early). Clarify, clarify -- no regrades on exam!
No office hours tomorrow.
Double-check re-grades, HW, etc. Can't fix problems after the 11th.
Wiki.
Thanks!
- draw phenotype & genotype w/ arrows
- forward: no prior genetic knowledge required
- forward: requires lots of mutagenesis and screening
- reverse: need to "know" locus
- only gets @ effect of 1 gene
Mutagenesis important for both approaches
- chemical -- EMS, high rate, usually hypomorph/hypermorph
- physical -- radiation, medium, usually loss of fxn, rearrangement
- biological -- TEs, low, can be loss of fxn in gene or hypomorph in reg.
Often make mutant "libraries" of lines of mouse or rice or whatever with known mutations. Broad use to community.
Identify mode of action -- crosses (either with Natural mutant or artificial one)
* why are dominant mutations rare? (most will be loss of function or hypomorphic)
* advantage of haploid organisms? (all mutations seen)
* do mutations complement? same vs. different.
* under what conditions does mutation have effect? gets at function
Map mutant * QTL mapping & fine mapping
Test candidate loci: do they have differences from WT (nonsyn etc.). Show expression diffs? (how measure? RNAseq)
Start with locus of interest. Want to know what it does.
What does sequence tell us? Important domains, shared promotoers, etc.
Mutagenize and look for fxn.
- knockout or knockdown (how do latter? RNAi)
Where/when is it expressed?
- RNAseq in different tissues.
- Transform promoter/enhancer w/ reporter gene (example? )
- in situ hybridization: label DNA/RNA and see in what tissues it hybridizes
Levels of protein:
- Western blots