should I design primers spanning both intron and exon - (Nov/20/2011 )
I hope to check if a gene is normal in patient. I plan to do PCR of an exon, and send for sequencing.
My question is, should I design the primers in intron, in exon, or spanning intron and exon? How should I choose among them?
Thanks a lot!
Do you mean you want to know if there are mutations of the gene in patients? If yes, you can just sequence exons. You can design primers on introns to span exons.
Design the amplicon so you cover the whole exon (depending on the size, if it's too big you need to design more amplicons) plus some 50 bases of introns on each side, so you cover the whole exon in sequencing read(s). Ask for the usual read length of your sequencing facility to know how big amplicon you can have.