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Lab 3: Forward Genetics Project: Linkage Analysis
Now that you have found from your mutant hunt a visible marker of a gene defect (phenotype alteration from wild type), you will determine on which of the five autosomes (linkage groups) the gene mutation responsible for your aberrant phenotype is located. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked mutations will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas linked mutations will not.
In practice, linkage tests are performed using the following steps (where "d" (dpy) represents your recessive mutant tested with reference marker "u" (unc)). The markers d and u must be visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your dpy mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation unc(+/+; u/u) and have no dpy mutation. The genotypes of the F1 hybrids will be (+/d;u/+) and (+/+;u/+). We are only interested in the double heterozygote (+/d;u/+). The F1 hybrids containing only u are not useful. To select the (+/d;u/+) heterozygotes, we let 4 to 5 individual F1's self fertilize on separate plates (one animal on each plate). We score the progeny of the F1 individuals (the F2) for linkage. Only F1 worms which produce d/d homozygotes are scored, since those are the (+/d;u/+). You should find d/d homozygotes on 50% of the plates. Why?
F2 progeny of each class are counted in the (+/d;u/+) plates: wild-type (+/+;+/+); d (d/d;+/+); u (+/+;u/u) and du double (d/d;u/u). If assortment is independent, progeny will be: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!
On the other hand, if the markers are closely linked double homozygotes (d u/d u) will occur only through a very rare recombination event; therefore, you are not likely to observe the double mutant class.
To Do Today:
1. For linkage testing set up four different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate.
2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 5 known Unc (+/+; u/u) mutations on a mating plate. The strains and their reference mutations are:
| Chromosome || Strain || Phenotype
| Chromosome 1 || unc-13 || coiler
| Chromosome 2 || unc-52 || immobile
| Chromosome 3 || unc-32 || coiler
| Chromosome 4 || unc-17 || coiler
| Chromosome 5 || unc-60 || immobile
3. Label your five plates with your PURPLE Sharpie. With the genotype of the strain - for example: +/+; unc-13/unc-13 (H) X d/+; +/+ (M) with your initials and date.
4. Incubate all of the worms at 23°C for 3 days in your team's worm box.
3-4 days after lab:
- For linkage testing, transfer 2 wild type cross progeny (heterozygous for both traits) that are L4 stage hermaphrodites from each of your 5 crosses to each of 2 new plates per cross for a total of 10 plates.
- Label your 10 (5 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
- Incubate all worms at 23°C until the next lab period.
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
Links to Labs& Project Info
Lab 11: RT PCR reactions
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Mapping Part 2
Lab 6: Score & DNA sequencing analysis
Schedule of Reverse Genetics Project
RNAi General Information
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification