I´m using High Resolution Melting (HRM) approach (Roche) for the gene screening of a gene with 4 exons. I have got fine results for exons 2-4 but i can´t get the result for exon 1. I´m doing all the same like i did with other 3 exons but in each run there is a very low amplification and the increase in the fluorescence signal is very late (after cycle 37). That makes impossible to distinguish samples that have mutations since they are recognized by software either as a non template samples or there is too many samples group based on their Tm.
So far, i have tried to optimize reaction with classic PCR troubleshooting (MgCl2, annealing Tm...) and i have also tried another primer for the same exon but the result wasn´t any better. I have also made the parallel runs of the same samples with different primer combinations at the same run,and i got result for the other exons but not for this problematic first exon again.
These primers that i´m using have additional small part of M13 sequence (other colleagues are using them for the sequencing),so i would like to ask if that can somehow affect HRM reaction and should i perhaps use classical PCR primers instead.
I´m kinda running out of ideas what could be a problem with this problematic first exon so any advice or idea what could be wrong or if you experienced the similar problem in the past, would be very useful.
Many thanks for your help.