PCR Primer Design

Question

[ebaduareb001]

I attempted to design primers for detecting an INDEL in a specific gene. I used the variant table on Ensembl.org to select variant IDs (rs numbers) and the location of the INDEL, focusing on loci with alleles longer than 8bp. For each chosen location, I extracted a 1200bp sequence from the target gene—600bp upstream and 600bp downstream of the INDEL. However, none of the primers designed using this 1200bp sequence successfully detected polymorphisms. Could the issue be with my procedure, or is it possible that the locus simply lacks polymorphism?

[ATpoint]

This is impossible to answer. All you essentially say is "my cake does not look like in the cookbook, but I don't know why". You need to add details, for example the evidence that the INDEL exists. Some IGV screenshot for example.

[ebaduareb001]

Thank you for the response. My question is how to design a primer for INDEL.

[ATpoint]

The same as any primer. Using software such as PrimerBLAST to ensure your primer is specific, follows the established heuristics towards length, GC content, GC clamp, no self-annealing, melting temperature, and that it does not bind elsewhere to produce a similar-sized product.

[ebaduareb001]

Which sequence may I use? or how can I know the location of the INDEL in target gene?

[ebaduareb001]