Hello, I want to do sanger sequencing for 3 SNPs in the CETP gene. Two of them are located in exons and one of them in intron. What should I consider for designing primers?
Make sure the amplified sequence contains all of the SNPS, or in a few products as possible.
Also, know that the first and last bp of a sequencing run are low-quality (50-100bp depending on your machine). Make sure the SNP is at least 100 bp away from your primer.
Finally, you can use more than one primer to sequence the same amplified PCR product. I'd recommend reading from both directions (forward & reverse), if nothing else to verify the SNP.
Good luck!
Adding to Dr. Katie's point, it is also be recommended to inspect SNPs at the primer-binding site to avoid any primer mismatches.
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