I decided to amplify my desired gene sequence for some strains of my sample and after that study polymorphism but my gene sequence have a high identity to another gene so I can, t design primers for full length gene amplification. What can I do? Do you think I can use HRM technology and design suitable primers for HRM? What do you suggest ? Thank you all.
Hi Mahboubeh
With HRM technique you can also study polymorphisms, but the important point in this technique is ‘primer’. They should be designed professionally. As I know, in polymorphism study the whole gene length amplification is not necessary. Anyway, I can’t suggest you a good way, because I don’t know the gene characteristics and also the kind of polymorphisms (e.g. SNP, insertion,…).you should negotiate with a person who notified about the detailed and design the best primers.
Good luck
You can use HRM to identify polymorphisms but if your gene of interest has strong homology to another gene or a pseudogene you need specific primer for the area of interest, irrespective of the method of polymorphism detection.
Yes, you can try the using of HRM to identify polymorphisms, but you need specific primer due to the high homology of your gene sequence with onother one. You could also try the of fluorescence resonance energy transfer (FRET) primers for identifying SNPs.
Surely, you already know this, but the better ways for HRM primer design are the choise of a target region length of
I agree with the other contributors to your post, it is possible to use HRM to screen for polymorphisms, however the process is primer dependent. If you are having problems with high sequence homology to another gene/pseudogene, you could consider using Locked Nucleic Acid LNA oligos which are template specific. I would also recommend PCR amplicons around 100 bp long for HRM to reliably detect melt temperature shifts. That said we have successfully detected SNPs with 300 bp long PCR products.
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