Agrogene demonstrated a simple procedure to validate SNPs. We are not aware of any IP on this method (beyond PCR) and it is the simplest method we could come up with. Briefly,
Examine the sequences of a contig with a viewer. Identify SNPs and also bases that are specific to one particular homoeologue locus.
Design a primer with a tm of 60 that has as its 3' end the base specific to the homoeologue locus (= Locus primer. This will enable the amplification of one locus, instead of the three or more that may be present).
Design two alternative primers to detect the SNP (one for each base known to be present at the site), each with their 3' end on the SNP (SNP primers), but make these primers different lengths (say 20 and 23 bases). The primers can be extended at their 5' ends by adding bases encoded in the DNA or for example adding a few Ts.
PCR with the locus primers and SNP primers will generate bands that have one of two sizes, according to which SNP primer was incorporated, and hence indicating the base in the original templates.
We recommend the tm calculation as performed by Applied Biosystems Primer Express, and that tms should be in the range 58-61. In our experience a touchdown PCR of 3 cycles per degree, from 60-55 anneal , then cycle at 55C anneal, tends to generate OK results, but we may revise our opinion after more experience. In order to score the products on sequencing gels the PCR product should ideally be < 400 bases.
Additional details and explanation
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