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PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans   

Materials and Reagents

1. PK lysis buffer
   
2. Proteinase K (Sigma-Aldrich, catalog number: P6556 )
   
3. Common PCR reagent (e.g., Invitrogen PCR kit – Life Technologies, Invitrogen™, catalog number: 10342-020 ; or home-made Taq and buffer)
   
4. Restriction enzymes (NEB)
   
5. Agarose gel
   
6. Ethidium bromide (Life Technologies, Invitrogen™, catalog number: 15585-011 )
7. Plus DNA Ladder (Life Technologies, Invitrogen™, catalog number: 10787-018 )
   

Equipment

1. MJ Research PTC-200 Thermo Cycler (MJ Research)
   
2. Thin-wall PCR tubes (USA Scientific, catalog numbers: 1402-2700 or 1405-8100 )
   

Procedure

1. Isolate genomic DNA with proteinase K digest.
   Tip 1. Typically, a large amount of PK lysis buffer is prepared (for the recipe, please refer to Caenorhabditis elegans/DNA/Single worm PCR) with supplement of proteinase K, and then small aliquots are stored (e.g., 1 ml) at -20 °C. This robust enzyme works well at a range from 20 μg/ml to 100 μg/ml, and the key is to activate it at 60 °C for ~1 h and then kill it at 95 °C for ~15 min or so. You do not want to see proteinase K torture your Taq enzyme during the subsequent PCR reaction.
   Tip 2. I prefer to pick reasonable numbers (e.g, 10) of gravid adult animals and stick them into a PCR tube with PK lysis buffer (e.g, 20 μl). It does not hurt to use more than 1 worm per PCR reaction (genomic DNA from ~1/2 worm works just fine for most robust PCR genotyping). For PCR-RFLP, it’d be better to use more than 1 worm per reaction (e.g., 5 to 10). However, too much DNA template, in some cases, may inhibit your PCR reactions.
   
   
2. Perform a standard PCR, 20 μl per reaction.
   1. Set up the PCR, by adding the following component into a thin-wall PCR tube on ice in this order:

      DNAase-free ddH2O	11.0 μl
      dNTP mix (10 mM each)	0.4 μl (final, 200 μM each)
      Forward primer (10 μM)	0.4 μl (final, 0.2 μM each)
      Reverse primer (10 μM)	0.4 μl (final, 0.2 μM each)
      PCR buffer (10x)	2.0 μl (final, 1x)
      MgCl2 (50 mM)	0.8 μl (final, 2.0 mM)
      Worm lysates (20 μl)	2.0 μl
      Taq (5 U/μl)	0.1 μl (final, 0.5 U per reaction)

   2. Run PCR (put the tube on the block when it is hot):

      1 cycle	94 °C, 3 min
      30 cycles	94 °C, 10 sec; 58 °C, 30 sec; 72 °C, 30 sec
      1 cycle	72 °C, 10 min

      
      
3. Digest with respective enzymes, 37 °C, O/N. Prepare multiplex (N+2) for N reactions:

   ddH2O	2.5 μl
   10x buffer	2.5 μl
   Enzyme (5 U/μl to 20 U/μl)	0.2 μl

   Aliquot 5.0 μl into each PCR tube.
   
   
4. Resolve O/N digest of PCR products on 2.0%-2.5% agarose gel.
   For daf-2(1370):
   Bands expected from Nco I-digest (NEB buffer 3): Wild-type, 202-bp; mutation, 234-bp.
   For daf-2(1368):
   Bands expected from Acl I-digest (NEB buffer 4 + BSA): Wild-type, 215-bp; mutation, 186-bp.
   Tip 3. Due to the small difference between the sizes of products, it is recommended to run the gel for a long time. Always add wild-type and mutants with known genotypes as controls. Ethidium bromide migrates toward cathode (-), just the opposite to the direction of DNA migration. To help subsequent visualization of DNA under UV light, it would be advised to add a few microliter of ethidium bromide (10 mg/ml) into the electrophoresis buffer near the anode (+).
   

Representative data

Figure 1. Representative data of PCR genotyping are shown here. 20 μl of daf-2(e1370) allele-genotyping PCR products were digested with Nco I at 37 ºC overnight. The digested DNA fragments were resolved on a 2.0% agarose gel. Expected sizes of DNA bands: wild-type, 202-bp; daf-2(e1370) mutation, 234-bp. Lane 1: 1 Kb Plus DNA Ladder. Lane 2, 7, 8: daf-2(e1370)^+/+; Lane 4, 5: daf-2(e1370)^+/-; Lane 3, 6: daf-2(e1370)^-/-. The results are highly reproducible, and necessary controls should always be included to assure the results.

Recipes

1. PK lysis buffer
   10 mM Tris-HCl (pH 8.0)
   50 mM KCl
   2.5 mM MgCl₂
   0.45% Tween-20
   0.05% gelatin
   20 g/ml proteinase K
   Prepare and store small aliquots at -20 °C
   

Acknowledgments

This protocol was adapted from work performed by members of the Kenyon lab, including PZ. PZ was supported by a postdoctoral fellowship from the Larry Hillblom Foundation.

References

1. Gems, D., Sutton, A. J., Sundermeyer, M. L., Albert, P. S., King, K. V., Edgley, M. L., Larsen, P. L. and Riddle, D. L. (1998). Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics 150(1): 129-155.
   
2. Neff, M. M., Turk, E. and Kalishman, M. (2002). Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18(12): 613-615.