Primer design for SNP detection - (May/29/2008 )
Some homework over the semester.
I have been tasked to design a set of primer to detect the presence for a single nucleotide polymorphism (SNP)..Well, it is on the p53 gene and i got my control primers to work.
But something is wrong, my polymorphic primers actually managed to amplify something which shouldn't happen in the first place as this SNP is very rare in general population and I am using HCT116 cell line which should not have the SNP.
Well, my professor told me that the last nucleotide of the primer must be the same nucleotide as the polymorphism and so, if there is indeed a SNP, the primer would prime onto it. Is this the correct basics of designing a primer in my case?
I can provide the primers sequence here if it is needed.
I have been tasked to design a set of primer to detect the presence for a single nucleotide polymorphism (SNP)..Well, it is on the p53 gene and i got my control primers to work.
But something is wrong, my polymorphic primers actually managed to amplify something which shouldn't happen in the first place as this SNP is very rare in general population and I am using HCT116 cell line which should not have the SNP.
Well, my professor told me that the last nucleotide of the primer must be the same nucleotide as the polymorphism and so, if there is indeed a SNP, the primer would prime onto it. Is this the correct basics of designing a primer in my case?
I can provide the primers sequence here if it is needed.
Your prof is right. Having the variant nucleotide at the 3' end means only those molecules with the SNP will amplify.
For a SNP, there are at least two variants. You should design two pairs of primers. They share a common upstream or downstream primer. ONe pair will only amplify one variant. So the two pairs can mutually prove each other. This type of PCR is called allele-specific PCR. It does sometimes give nonspecific amplification. I prefer restriction digestion after PCR.