ExonPrimer is a Perl script that helps to design intronic primers for the PCR amplification of exons. The script needs a cDNA and the corresponding genomic sequence as input. It aligns these sequences using Blat and designs PCR primers to amplify each exon using Primer3. The positions of the exons are deduced from the alignment of the genomic and the cDNA sequences. Insertions/deletions up to 6 base pairs are bridged by postprocessing. Exons with small introns in-between are combined. Exons smaller than 20-25 bp will not be recognized. The user can define the maximum exon size. Exons larger than this size will be divided into several parts.
The poly-A tail of the cDNA should be clipped to allow the alignment of the cDNA and the genomic DNA sequence. The genomic sequence must be longer than the cDNA sequence. Otherwise the design of primers for the first and/or last exon is not possible.
Download of the human genome sequence with all SNPs masked by N's. Using this sequence, one can avoid primers to be positioned across SNPs.
ExonPrimer is also available in the UCSC Genome Browser for the human and mouse genomes. One can find the link to ExonPrimer in the 'Sequence and Links to Tools and Databases' section of the UCSC genes details page.

The exons and 600 base pairs upstream and downstream of the exons will be used for a Blat search against the entire genome. A score of 300 means that all hits with more than 300 identical base pairs will be masked. Higher scores will result in masking only duplicated regions. Lower scores will also mask repeats.

An EMBL formatted sequence file will be generated containing exons and intron/exon boundaries. The sequence can be imported as reference sequence into the Staden package. If 'CDS start' and 'CDS end' are empty, the coding sequence will extend from position 1 up to the end.
CDS start    CDS end    Maximal intron length