Help me w/me homework - Assay Design (Nov/29/2008 )
I'm just learning this molecular stuff, but I need help with this assignment: I must design a test around a gene that is provided to me (240 bp long starting at 301 and continuing to 541) and the polymorphism (y) is at 407. I know there are different assays to use, but what would be the best? I'm trying to construct primers for this, and I'm having a terrible time - I must be doing something wrong. LL
First of all, the best forum for this would be the homework forum, and second, what assays do you know about? Is the polymorphism a SNP or is it larger than that? The answer to that question will affect what assays you can use to detect the polymorphism.
Primers are a good start for several assays though!
If you know what the sequence of the gene is, then I would first do a restiction digest analysis (by computer) to see if there are any restriction sites that change due to the polymorphism. If so, then you would design primers at the ends of the gene, and do a restiction digest to identify the mutation in whatever samples you have. If there are no changes, then I would just design primers to make a PCR product to sequence. I know that there are ways to identify polymorphisms by real time PCR, but this method would be most reliable in my opinion.