I want to design some primers to characterize differents SNP (Single Nucleotid Polymorphisme) in a simple process using PCR and agarose gel, not for sequencing. What could be the appropriate way to design them. Should I place the changed nucleotid (SNP) inside the primer's sequence or at the end 3' ?
You cannot detect a SNP on an agarose gel. You need to either add a restriction enzyme, assuming the SNP changes a restriction site, or use high-resolution melting.
Your primers should surround the region with the SNP, but NOT include the SNP in the primer region or you will simply replicate your primer in all of your products and you will not be able to detect the SNP.
Please see:
Guo-Qiang Jiang et al 2013
A Simple CELI Endonuclease-Based Protocol for Genotyping both SNPs and InDels
You cannot detect a SNP on an agarose gel. You need to either add a restriction enzyme, assuming the SNP changes a restriction site, or use high-resolution melting.
Your primers should surround the region with the SNP, but NOT include the SNP in the primer region or you will simply replicate your primer in all of your products and you will not be able to detect the SNP.
look up dcaps on google. it is a method for adding a deliberate mismatch into a pcr prime and it designs the primer for your. in conjunction with the naturally occurring snp it will produce a restriction enzyme cutting site over 98% of the time.then just amplify a short (150 base) amplimer and cut with the enzyme, if it cuts one allele is present but not the other, then the fragments 150 or 125 define the polymorphism present
also consider using ARMS primers to specifically amplify each base separately
You have to add restriction enzyme after PCR process to detect SNP chnges on an agarose gel and for this you need to use high-resolution melting, like overnight or atleast for hour incubation process. It will be written on your catalog of restriction enzyme.
Your primers should be around your SNP, dont include the SNP in the primer region otherwise you will not be able to detect the SNP changes.
And i am agree with Dr. Katie A Clark
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology