by Michael Koelle, adapted from R. Barstead and Williams et al., Genetics 131: 609-624, 1992.
4/6/94
Introduction: This method is used to establish linkage of a mutation to a particular chromosome cluster or arm. Its advantage over mapping with visible marker mutations is that you can test linkage to all the chromosomes simultaneously without having so many marker mutations present that the animals are sick and confusing to score. Also, it saves a generation, so it's faster.
Although the original paper suggests continuing to use STS polymorphisms to place mutations in smaller chromosomal subregions, most people opt instead to use 3-factor mapping with visible marker mutations for this finer level mapping. The 3-factor method allows you to select and analyze only those animals which are products of informative recombination events, whereas the STS polymorphism method does not. Therefore, as you progress to higher levels of resolution, the STS method forces you to analyze large numbers of mostly uninformative animals. Also, 3-factor mapping with visible markers generates as a by-product double mutant chromosomes in which your mutation is cis-linked to various marker mutations. Such cis double mutants are very useful, for example, in setting up complementation tests against other mutants, in strain constructions, and in further mapping experiments.
Strategy: The mutant of interest is mated to the strain RW7000, a derivative of the wild-type Bergerac strain which carries a large number of TC1 transposons that are not found in the normal laboratory strain N2. In the case of a recessive mutation, 12 or more homozygous mutant progeny are picked from the mutant/RW7000 heterozygotes. PCR is used to analyze the DNA from each individual worm for the presence or absence of individual RW7000 TC1 inserts found on each of the chromosomes. In the case of a TC1 that is unlinked to the gene being mapped, about 3/4 of the animals will carry the TC1 (either heterozygous or homozygous). In contrast, a linked TC1 will be detected in significantly fewer of the animals, since this would require recombination between the mutation and the TC1. The TC1s are detected by PCR using one common primer that hybridizes to TC1 and points outward toward the flanking C. elegans DNA, and one specific primer that hybridizes to genomic DNA flanking a particular RW7000 TC1 insertion site. These specific primers have been designed such that the PCR product from each TC1 insertion is of a different size. Thus up to about 6 of these specific primers can be included in a single PCR reaction, and the various PCR products can be resolved on an acrylamide gel. This allows you to test the linkage of your mutation to all the chromosomes simultaneously by analyzing only about 12 worms.
Mapping a recessive mutation
Mapping a dominant mutation: RW7000/mutant heterozygotes are generated, as described above, but non-mutant progeny of the heterozygotes are picked and analyzed for STS polymorphisms. In the case of a TC1 that is unlinked to the gene being mapped, about 3/4 of the animals will carry the TC1. In contrast, a linked TC1 will be detected in nearly 100% of the animals. While the difference between ~75% and ~100% may seem annoyingly small, forcing you to analyze a large number of worms to get a significant result, this mapping method actually works better than you might expect at first thought. Because both the mutation being mapped and the STS polymorphisms are in effect dominant, this scheme exaggerates linkage; recombination between the mutation and a linked STS polymorphism will only be detected if it occurs in both the sperm and the egg that generate the animal being analyzed. Thus you can truly expect very close to 100% of the animals to carry the linked STS polymorphism; for example even if the STS polymorphism and the dominant mutant are 20 map units apart, 96% of the animals analyzed will carry this linked STS polymorphism.
Mapping a dominant mutation
1. Crosses: Note that RW7000 is a somewhat sickly strain. Males will not mate, and the hermaphrodites are sterile when grown above 20deg.. Therefore the cross is set up by mating mutant males to RW7000 hermaphrodites at 20deg.. If mutant males cannot mate, then mutant/+ males (generated by mating N2 males to mutant homozygotes) can be used.
2. Pick all the adults to be analyzed by PCR to one unseeded plate (letting them crawl around on the new plate allows you be certain you have separated them from any eggs or L1s). You may want to pick enough worms so that if your mutation turns out not to be tightly linked to any of the chromosome clusters (which is what you will test first), you will already have worms stored away in the freezer to test for linkage to STS markers on the chromosome arms. Now transfer individual worms with a worm pick to a 2.5 ul drop of worm lysis buffer previously aliquoted into the cap of a 0.5 ml microfuge tube (tube still attached). Watch the transfer under the dissecting scope, and try not to transfer too much bacteria. Close the tube and leave it inverted (it is convenient to place the upside down tubes in the holes of a styrofoam 15 ml centrifuge tube rack) while you pick the rest of the worms. Next the worm/lysis buffer are deposited in the bottom of the tube by a brief microfuge spin, and the tubes are frozen at -70deg. for >10 minutes.
3. The tubes are thawed, and 2 drops of mineral oil (from a blue tip) are placed in each tube. Tubes are placed in a PCR machine programmed to heat to 60deg. for 1 hour, followed by 95deg. for 15 minutes.
4. At the end of this time, 22.5 ul of PCR "master mix" is added to each tube, and spun down through the oil by briefly microfuging. The tubes are returned to the PCR machine and amplification is immediately started using the following parameters: (94deg. 30 seconds; 58deg. 1 minute; 72deg. 1 minute) repeat for 30 cycles; 72deg. 5 minutes; hold at 4deg..
5. Half each reaction is loaded on a 0.8 mm thick 6% acrylamide gel, and electrophoresed until the bromophenol blue marked has migrated about 3 inches. A convenient method to load the gel is to place a number of drops of loading buffer on a piece of parafilm, and to then use a P20 pipetteman to withdraw the sample to be loaded from the tube, to mix it with a loading buffer drop on the parafilm, and then to load it on the gel. Any mineral oil will remain stuck to the parafilm.
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Solutions:
Worm lysis buffer (filter sterilize and store at room temp., or freeze)
50 mM KCl
10 mM Tris pH 8.2
2.5 mM MgCl2
0.45% NP-40
0.45% Tween 20
0.01% gelatin
Just before use add 6 ul 10 mg/ml proteinase K (in water,store in frozen aliquots) per ml of lysis buffer.
PCR reaction conditions
2.5 ul of worm lysis buffer conaining a worm
20 picomoles of each primer
2.5 ul 10X PCR buffer (100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin)
4 ul of dNTP mix (1.25 mu for each dNTP, stored frozen)
0.125 ul of Taq polymerase (5 U/ul stock)
H20 to 25 ul
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Primers:
The standard set of primers for testing linkage to the clusters on each of the chromosomes are listed below, with the size of the PCR product generated. See Williams et al., for additional STS markers for the chromosome arms.
Polymorphism
name chromosome product size (bp) primer sequence
maP1 II 234 CCA ATT TTC CGG AAG TTT TCG
stP103 X 209 GAC GAA AAG AGG TAC ACG AG
sP4 IV 179 TTT CTG TTT TGT GCT TAG ACG
mgP21 III 165 GGA ACA AAA GTG CCT TGG G
hP4 I 130 CGG AAA TAT TAT CAG CAC AGC
bP1 V 119 AAC ACA TTT AGG TAA TGT AGC AC
---------- The common TC1 primer "618" GAA CAC TGT GGT GAA GTT TC
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Notes:
1. Showing linkage to the X chromosome actually doesn't require any markers at all, since transmission of the mutation along with the X chromosome through males may become obvious during the two generations used to generate the worms for PCR. However, following through with the STS mapping can show that the mutation is not linked to the autosomes, and therefore behaves like a single locus.
2. If the mutation being mapped is embryonic lethal, PCR reactions need to be carried out on single mutant dead eggs. To do this: using a drawn out 5 ul capillary tube, place a small amount of chitinase solution onto the dead egg, and immediately pick up the egg and transfer it to 2.5 ul of worm lysis buffer in a tube cap, as described above, taking care not to transfer too much chitinase solution. Then proceed as described above.
Chitinase solution:
20 mg/ml chitinase (Sigma # C-6137)
50 mu NaCl
70 mM KCl
2.5 mM MgCl2
2.5 mM CaCl2
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