Lecture 8

Steps of genetic analysis

1. Define the problem: Let the question guide your analysis, not the other way around

2. Choose an organism: Consider mutation rates, number of organisms needed, space they take up, etc.

3. Perform a mutant hunt: You want to find an animal that has a phenotype of interest.

   • Consider mutagenesis to increase chances of finding a loss-of-function mutation (10^-3 with mutagenesis) or a specific mutation (10^-5 - 10^-6) with mutagenesis
   • Perform a screen or a selection experiment to isolate mutants of interest

4. Screen until saturation: Keep in mind limits of time and number of mutations you can expect to find

5. Establish a strain: You need a strain that is viable and fertile. If you are propagating a heterozygous strain (usually if homozygous mutants are lethal), use a balancer if possible.

6. Backcross and/or outcross: To remove background mutations - Backcross to the parental strain used in the screen/selection. Outcross to a wild-type strain.

7. Test for dominance: By generating heterozygous strains and determining if they display the mutant or wild-type phenotype.

8. Single-gene phenotype: Determine if the mutation is caused by an allele of a single gene.

9. Mapping and complementation: To identify where in the genome the gene of interest lies.

10. Characterize the phenotype: Look at your mutants in detail. What part of which process has been modified? Is this a pleiotropic locus (one locus that impacts multiple traits)?

11. Define the nature of the mutant allele: Is the mutant a hyper/hypomorph? How many copies of the mutant allele are required for the mutant phenotype?

12. Perform non-complementation screens: Find new mutations that fail to complement the original mutant phenotype.

13. Define the null phenotype: Make using genome editing, screen for independent loss-of-function alleles (early stop codons, frameshifts), or generate a deletion that removes the coding sequence.

14. Clone the gene: By complementation, phenocopy, or sequence.

15. Determine where the gene is expressed: Using in situ hybridization, antibodies, fluorescent transgenes.

16. Determine site of gene action: Rescue mutant phenotype in a single cell/tissue or cell autonomy experiments.

17. Determine the time of gene action: By inducing expression at a specific time or by generating temperature-sensitive mutants and testing them at different temperatures.

18. Determine if there is maternal/cytoplasmic inheritance: This requires observation through several generations after crossing.

19. Determine the over expression phenotype: By expressing multiple copies of your gene.

20. Perform an overexpression screen for additional modifiers: Screen for dominant phenotypes similar to your mutants, inducible overexpression, transposon-mediated overexpression.

21. Isolate suppressors and enhancers

22. Investigate pathways: Interactions and epistasis