I want to use a primer extension assay to distinguish between a mutated and wild-type sequence using qPCR.
What polymerase would be ideal for such an assay? I need something that stops dead in its tracks when it encounters a mutation or a block thus preventing primer extension and amplification completely in the mutated sequence.
What temperatures should I be looking at with respect to the Tm of the primers to conduct such an assay?
Where should the primers ideally be on the sequence with respect to the mutation-upstream to the mutation, on the mutation?
Is there any specific product that you would recommend?
The type of primers that you are describing are used in Tetra ARMS-PCR, but they usually know at what position the polymorphism is so they can set it as the last position in the forward primer.
Can you please provide more details for the ARMS pcr method? How are primers designed? I was hoping for like an allele specific -qPCR assay with 2 primers and a probe. Is this feasible?
If you anyway use a probe, then I would not go for an allele-specific PCR but rather use the probe to distinguish the sequences. What kind of probe are you using?
To enhance the specificity of your amplification I recommend a TMAC buffer :
http://www.ncbi.nlm.nih.gov/pubmed/10871414
http://www.ncbi.nlm.nih.gov/pubmed/18281424
Tetra ARMs PCR is a multiplex assay where you have a forward primer going in one 5'-3' direction that is specific for one of your SNPs in the last primer nucleotide, and there is another antisense forward primer that is specific to the other form of the SNP coming from the other 5'-3' direction. Usually they design the assay so reverse primers are after different lengths so you can visualize different sized bands for each SNP on a gel electrophoresis.
While this is a pretty nifty idea, if you've already got a qPCR machine with high-resolution filters, I'd suggest looking into High-Resolution Melting Analysis (HRM or HRMA). This is not a primer extension assay, but it is a much easier diagnostic tool to use, and they sell cheap premade kits. You just make a common primer set that would work on both SNPs and the machine can tell you if you have an SNP based on a dissociation curve from your melts.
If your machine doesn't have high resolution filters I would also look into Molecular beaconing or TaqMan probes.
Thanks,
Dave
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