Abstract
This protocol describes the basic principle of PCR/restriction digest genotyping of point mutations in worms, based on the principle of Restriction Fragment Length Polymorphism (RFLP) analysis. This type of genotyping is, particularly, useful when phenotypic analysis of animals carrying point mutations is difficult (e.g., in a complex genetic background). I will illustrate the general procedures, using an example of daf-2 gene, encoding the sole insulin/IGF-1 receptor of C. elegans. Gems et al.(1998) did a very elegant job and characterized a series of mutations of daf-2, including the following two temperature-sensitive hypomorphic alleles: daf-2(e1370): Substitution C/T (wild type/mutant), amino acid change: Missense P to SFlanking sequences: 5’-CTCTATGAAATGGTTACACTCGGTGCTCAGCATATATTGGTTTGAGTAATGAGGTGTIntracellular kinase domain, Class I, strong phenotype. daf-2(e1368): Substitution C/T (wild type/mutant), amino acid change: Missense S to LFlanking sequences: 5’-TCCGGAATTTACGTATTGAGGCAAAGTACTGTTCAGAAATVTATATGCTATCACAGTExtracellular ligand binding domain, Class II, weak phenotype. Here I will show you how to design the primers for PCR-RFLP analysis. daf-2(e1370): Designed by Seung-Jae Lee from the Kenyon labForward primer: 5’-CGGGATGAGACTGTCAAGATTGGAGATTTCGG-3’Reverse primer: 5’-CAACACCTCATCATTACTCAAACCAATCCATG-3’On the (-) strand, the nucleotide next to the 3’ end of reverse primer is G in wild-type allele, which is mutated to T in daf-2(e1370). Thus, by introducing another mutation (double C here, highlighted) into the reverse primer, it creates an Nco I-restriction site (i.e., CCATGG) only for PCR products derived from wild-type but NOT daf-2(1370). daf-2(e1368): Designed by Peichuan Zhang from the Kenyon labForward primer: 5’-GTTCCGGAATTTACGACGTATTGAGGCAACG-3’Reverse primer: 5’-CTATCGGATCGAGTGGTATATTTAAC-3’Similarly, on the (+) strand, the nucleotides next to the 3’ end of forward primer are TC in wild-type allele, and TT in daf-2(e1368). Thus, by introducing another mutation (C here, highlighted) into the forward primer, a restriction site of Acl I (i.e., AACGTT) is generated in the presence of daf-2(1368) point mutation. The key is to introduce new mutation(s) at the 3’ end of one of your primers. Since the difference of the sizes of digest products is just ~30-bp, the length of the primer, you have to pick the other primer to generate an amplicon of ~200-bp – 250-bp or so. Here is a website that can help you design the primers with appropriate restriction site for genotyping: http://helix.wustl.edu/dcaps/dcaps.html (dCAPS Finder 2.0) (Neff et al., 2002).
Keywords: C. elegans, PCR genotyping, Point mutation
Materials and Reagents
Equipment
Procedure
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
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
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi Gloria,Sorry about the late reply. Thank you for your support to Bio-protocol and we will keep our efforts to bring more high-quality protocols to the users.Best,Peichuan
Hi Sophia,Thanks! I'm glad that our protocols have attracted attention from potential users.We have been using regular agarose gel (2.0%) and 1X TAE buffer all the time. In my case, I need to separate two bands of about 200-bp (with difference of ~30-bp), and sometimes, I used 2.5% agarose gel instead. Try to not use too high voltage to avoid "smiling effects", and always remember to have positive and negative controls for comparison. Also use fresh 1X TAE buffer, as its buffering capacity drops significantly after a few rounds. You may consider designing a few different PCR-restriction digest strategies and pick the one that produces best resolution.I only used TBE buffer, which is better than TAE in its buffering capacity, for gel shifting experiments. I recall that I saw some notes about achieving better resolution with TBE for smaller DNA (<300-bp on 2% agarose gel).Hope this would help you a bit. Please let me know if you have further questions, and best luck with your experiments.Peichuan
Hi Peichuan,Thank you so much for your detailed explanation! I'm going to try out your protocol and will let you know of my progress.Sophia
Hi Jimmy,Very glad that it worked out well for you. And thanks so very much for sharing your own experience, which is great and would definitely help us improve the protocol as provided on our website. We hope to provide more high-quality author-validated protocols to share among our research community. I believe that you have taken right action for PCR, by using modified primer, touchdown strategy and DMSO, to improve amplification specificity/efficacy in your case. Most Taq enzymes, unless otherwise modified, do not possess 3'->5' proof-reading exonuclease activities. Please check the info that I have found from NEB http://www.neb.com/nebecomm/products/faqproductm0273.asp In my case of daf-2 genotyping, I could actually distinguish heterozygote from homozygote. I could tell about two bands for heterozygotes, with wild type and daf-2 homozygotes as controls. I also used just regular desalted primers for genotyping.BTW, to confirm the PCR genotyping results, you can also use regular Taq to amplify a PCR product (e.g., 500-bp or so) around the mutation, and then sequence the product with a primer. Best,Peichuan
Hi Jimmy,I used very typical recombinant Taq (e.g., Invitrogen 10342-020) and it worked pretty well in my hand (home-made Taq also worked). I would recommend you not to use Taq w/ high proof-reading capacity. The other thing that I have in mind is that you probably would like to re-design your primers. Put GC clamp at 5' end, but not more than 4 GC at the 3'-end. Treat them as primers for qPCR.I run the gel for rather a long time (put a little bit of EtBr in the buffer), with the aim to separate the two bands, which differ just in ~30-bp or so (I don't really bother to check the 30-bp band though). In my case, I can always check the phenotypic readout for daf-2 mutations, which is dauer formation. If you have concern about the genotyping, you should also sequence the PCR product and check certain phenotype that are associated with the mutation.Best luck,Peichuan