- Predict the chromosome goi-1 is on through analysis of the phenotypes of the F2 generation.
- Design and begin setting up crosses to confirm the chromosomal location of goi-1.
Linkage Analysis: Phenotypes of the F2 Generation
Last week you set up self-fertilizations of heterozygous hermaphrodites, all of which should carry the unc reference allele, and some of which should carry our mutation of interest.
TO DO:
- Scan all 20 plates of F2 progeny.
- Discard any plates that have male progeny. These progeny were likely produced because your hermaphrodite was older than L4 and had already mated with one of the males on the previous plate. Because there is no way to know which animal she mated with, we cannot be sure that these progeny are F2 generation.
- Discard any plates that show only the wild type and Unc phenotypes. These progeny were likely produced because the hermaphrodite you chose carried only goi-1(+), and did not carry the mutant goi-1(su219) allele and so do not express the mutant phenotype.
- Mark on the board how many remaining scorable crosses you have for each Unc reference strain. Scorable crosses should have no males, and should express the phenotype associated with our mutation. Your instructor will help the class distribute the plates evenly so that each group is scoring a comparable number of crosses.
- Begin scoring your crosses. For each cross, you should examine and score the phenotype of a random sample of 50-100 F2 adult worms. Here are some tips for scoring:
- It is important to move randomly about the plate, and once you identify a worm, commit to picking and flaming that worm to remove it from the plate so it is not counted twice.
- The mutant worms may be smaller and not move as well as the wild type worms. WT worms may be under counted because they are harder to "catch". Be careful not to skew your data in this way.
- Look around your plate to get a quick assessment of the population. Pick what you consider single mutants to a transfer plate and what you consider double mutants to another plate. Ask your lab partner and your instructor to check them before you go too far in the scoring process. The main challenge is to correctly differentiate single mutants of each single phenotype from double mutants. Students tend to under-count single mutants and over-count doubles when they are inexperienced.
- Record in your lab notebook the number of WT, Dpy, Unc, or Dpy Unc mutants by carefully assessing the phenotype as you remove each animal from the plate. Be sure to flame the pick after removing each worm.
- Record your data on the course-wide spreadsheet.
- Based on the phenotype data from this lab, on which autosome do you think our mutation is located?
Test Cross and Map Distance
Assuming that you have determined the linkage group of goi-1, we will continue working with that strain only. You now suspect that the two genes (goi-1 and the reference mutation unc) are on a particular autosome.
TO DO:
- With your partner, design a cross using the available worms listed above that will enable you to confirm that your expected unc reference gene is linked to our gene of interest.
- NOTE: This will require more than one generation of worms! Remember that homozygous mutant males are unable to mate. In your design, indicate the genotypes of the worms you will mate, the genotypes of the gametes they can produce, and the genotypes and phenotypes of the resulting progeny. What ratio of phenotypes would you expect to see as a result of independent assortment? How is this different from what occurs when genes are linked? What statistical test can you use to determine whether they are linked? If they are linked, how can you calculate the distance between the two genes?
- Have your instructor check your strategy before moving on.
- Pick 3 double mutant L4 hermaphrodites to each of 2 separate mating plates.
- Next, transfer 3-4 wild-type males onto a transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to each of the mating plates.
- Label your plates with your PURPLE Sharpie. Incubate your worms at 23C for 3 days.
To Do in 3-4 Days: Test Cross
In the last lab, you set up a cross to generate heterozygous male worms carrying mutations in goi-1 and in an unc reference strain in cis. Today you will set up the actual test cross to confirm that they are linked and to determine the distance between them.
TO DO:
- Pick 3-5 double mutant L4 hermaphrodites (goi-1(su219) unc(ref)/goi-1(su219) unc(ref)) to each of 2 separate mating plates.
- Next, transfer 3-4 heterozygous male progeny (goi-1(su219) unc(ref)/goi-1(+) unc(+) onto a transfer plate to allow them to dissociate from any eggs or young larvae, then transfer them to each of the mating plates.
- Label your crosses with your PURPLE Sharpie. Incubate your worms at 23C.