Genes are usually expressed as multiple
alternative transcripts as a result of alternative splicing of the pre-mRNA.
Pan primers amplify all transcripts so the forward and reverse primers are
designed to anneal to exons that are common to all transcripts (see figure
below).
In order to amplify different alternative
transcripts via PCR or real-time PCR, you may need to custom design your own
primers if what you need are not commercially available.
Assuming that you are working with a known
gene sequence and that you also know which alternative transcripts you want to
amplify, you will need to design at least one of your primers, either forward
or reverse, to span across adjacent exons.
For example:
