Dear all, Thank you for all the replies I received regarding methods for small scale SNP genotyping. It seems a common problem in evolution and ecology studies so I'll summarise the replies I got in this email. To save space I'll not acknowledge people individually (unless when quoting their entire emails) but thank you all so much for sending me the suggestions! The most obvious one is restriction enzyme (PCR-RFLP / CAPS): If the SNP is in a restriction site, you can simply PCR across it, digest, and run on a gel. http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechCAPS.shtml I was also recommended the following tool for selecting restriction enzymes for CAPS: http://pgrc.ipk-gatersleben.de/snp2caps/ If the SNP isn't in a restriction site, one may be engineered: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechDCAPS.shtml Apart from restriction digestion, the most popular methods are allele-specific PCR (AS-PCR) and related: two allele-specific primers can be designed to complement the SNP at the 3' end. There are many variations to this, e.g. labeling the two allele-specific primers with different fluorescent dyes; making the two primers different lengths; running two separate reactions with the reverse primers at different locations so each allele-specific product is of different length; etc. This could also be made medium-throughput: Multiplex SNP-SCALE: a cost-effective medium-throughput single nucleotide polymorphism genotyping method Kenta, T; Gratten, J; Haigh, NS, et al. (2008) MOLECULAR ECOLOGY RESOURCES 8 (6) 1230-1238 David Remington kindly shared some experience in primer design for AS-PCR: 'My strategy would be to use Primer3 or something similar to design the primers to ensure you get something as well-behaved as possible. You would use the sequence for containing one of the two versions of the SNP, fixing the location of the SNP-specific primer and letting the program pick the second primer. If the SNP-specific primer has problems (e.g. too much internal base pairing), you can try making the SNP-specific primer the reverse primer rather than forward. Then use the other SNP allele in the template sequence and try the corresponding primer with the new 3' base, and specify the 2nd primer the program found for the first version to see if that is still acceptable. This may get tricky, and you might have to try a bunch of combinations, different 5' starting points for the SNP-specific primer, etc. As a last resort, you can relax the program's parameters slightly and see if you get something workable. 'I usually try for Tm close to 60 degrees (the Primer3 default) and use a touchdown cycle starting annealing at 62 and stepping down to 55 or 52 over the first 7-10 cycles. That helps take care of the possibility of additional mismatch sites causing problems. If you need to pick less-than-optimal primers based on self-complementarity, etc, doing a hot start on your PCR can reduce problems with primer dimers. One nice thing about this approach is that you're not very limited in where the 2nd primer goes, since all you need to do is see the band on a gel. It could be 200 bases away or 1200. 'I haven't tried the following, but you could even use 2 different 2nd primers for the two different allele-specific reactions to give you different product lengths. Then you could mix them together after PCR and run in the same lane on the gel. It would essentially look like a length polymorphism.' And Pirmin Nietlisbach shared his own method: 'The discrimination among SNP states is achieved through the competition of two reverse primers with their 3'-end complementary to either SNP state and being of differing length. One advantage of this method is that it can be combined with microsatellite typing or typing of several SNPs in the same multiplex PCR. I am currently working on a manuscript on this method, which I probably could send you sometime next week if you are interested (let me know). I attach you a paper, that also used almost the same method. The method does need some optimization, but I am not aware of a method that does not (even sequencing may pose some problems and potentially require cloning, if heterozygotes are not obvious in the electropherogram). However, careful primer design should keep optimization time to a minimum. It worked well for me when I designed primers in a way that the reverse primers had a 1-2 °C lower melting temperature and higher concentration than the forward primer. 'This should keep competition between the two allele-specific reverse primers high. I did not introduce deliberate mismatches into the primer sequence as Li et al. did (attached paper). However, as I was working with Y chromosomes, I did not have to distinguish between homo- and heterozygous samples (although the electropherograms clearly suggest that it should have been well possible if the method would have been applied to diploid markers). 'I would pay most attention to having slightly lower melting temperatures for the competing primers, higher concentration for them, and, if you intend to multiplex them, very similar annealing temperature for the different SNPs. 'However, as the reverse primers can only be varied in length, but not where they bind, this can be difficult. I also don't know how well the different primer design softwares work (as the melting temperature calculation should be reliable).' Other options: SNuPe (Single Nucleotide Primer extensions), e.g. Seddon et al. (2005) SNPs in ecological and conservation studies: a test in the Scandinavian wolf population. Molecular Ecology The GE healthcare MegaBace-SNuPe system was used in this study. I personally have used ABI's SNaPshot system before, but the people I then worked with now suggest Sequenom (http://www.sequenom.com/) for smaller scale work. One reply I got also mentioned Sequenom, but said that the price break comes at around 20 SNPs or more. Amplifluor SNPs Genotyping System (http://www.flowgen.net/page019.asp) High resolution melt (can be fiddly, depends on your locus) If going commercial, I've been recommended the following companies: 'Kbioscience (based in the UK) do SNP typing at ~ £70 per locus setup plus 10-20p/genotype thereafter.' 'You may want to try the Genome Quebec Innovation Centre, in Montreal Quebec, Canada. i have used them in the past and they seem the cheapest. http://www.gqinnovationcenter.com/index.aspx ' Maureen Liu Postdoc Research Fellow School of Biology University of Nottingham University Park Nottingham, NG7 2RD UK telephone: +44 (0) 115 8230 354 fax: +44 (0) 115 8313 128 email: maureen.liu@nottingham.ac.uk Maureen.Liu@nottingham.ac.uk