Primer Design

RT-PCR amplification of a particular mRNA sequence requires two PCR primers that are specific for that mRNA sequence. The primer design should also allow differentiation between the amplified product of cDNA and an amplified product derived from contaminating genomic DNA. There are two approaches to designing the required primers (see figure below).

Make primers that anneal to sequences in exons on both sides of an intron (Panel 1). With these primers, any product amplified from genomic DNA will be much larger than a product amplified from intronless mRNA.

Make primers that span exon/exon boundaries on the mRNA (Panel 2). Such primers should not amplify genomic DNA.