Design a new gene specific forward primer (upstream to the existing forward primer) and then perform 1st round of touch-down PCR with 10-15 cycles. Now use this PCR product (after diluting) as template to perform nested PCR (preferably touchdown). This will certainly help to get a specific PCR product.

Thank you very much, I’ll definitely give it a try!

Hi Elena

since last exon of the gene is deleted(in homo or heterozygous state?), sequence of the mRNA is changed. Does this change induce a transcription rate change or abolish the completion of this translation?

since you know the sequence and deletion site, you can as kamal suggested design primers to directly sequence the targeted mRNA from cDNA sample. applying gel electrophoresis will show you if you have only one band (or 2 if it's heterozygous) at expected sizes. size selection by picking directly the band on agarose gel could also help you amplifying and sequencing.

fred

Hi Frederic,

Thank you for taking the time to answer.

I tried to obtain at least the wt band with this method, but nothing was visible (the patient is heterozygous for this mutation).

I don't know about changes in transcription rate or completion whatsoever, but I'm starting to suspect a possible underexpression of the normal allele and maybe a rearrangement in the mutated sequence.

Thank you again,

Elena

Hi Elena

picking the band on a resolution agarose gel (2-4%) could allow you to isolate the right sequence.

all the best for the followings

fred