primer design questions for confused - (Oct/26/2006 )
I am new to PCR and wonder if someone could tell me why you would choose to design primers within an exon rather than crossing intron exon bounderies. In desigining primers within an exon what would it look like on an agarose gel compared with primers designed across bounderires. Do you still end up with cDNA designing this way. Confused?
If you are talking about designing primers for RT-PCR, it is better to have at least one primer span an exon-intron boundary or have the two primers span one or more intron. In the first case, the primers won't amplify contaminating genomic DNA in your cDNA sample. In the second case, the band amplified from genomic DNA will be larger than that from cDNA so that you will know whether there is genomic DNA contamination.
Check this thread for a tutorial how to design RT-PCR primers.
You may want to check out the AlleleID software for primer design ON and ACROSS exon junctions. This way you may detect alternate splicing as well as presence or absence of a protein.