The PCR Suite: batch primer design
MJ van Baren 1, P Heutink2
1m.vanbaren@erasmusmc.nl, Erasmus MC Rotterdam; 2p.heutink@erasmusmc.nl, Erasmus MC Rotterdam
Primer design is an essential and usually time consuming step in many experiments. In order to automate this process, a number of programs have been designed, one of which is the Whitehead's Primer3, accessible via the Web (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi ). In these programs, parameters such as GC content, melting temperature and dimerisation parameters for the primers can be adjusted, and the best primerset for the sequence provided is calculated. Unfortunately, the input for those programs is usually a single sequence, generating one product. This makes them unsuitable for batch primer design.
We have created a new interface to the Whitehead's Primer3 program, allowing batch upload of sequences and design of overlapping PCR products. The PCR Suite consists of four interfaces: Overlapping Primers, Genomic Primers, SNP primers and cDNA primers.
The first program, Overlapping Primers, takes in a sequence and designs overlapping PCR products. The user can set the product size and the size of the overlap between products.
Genomic Primers and SNP Primers accept GenBank formatted files, extracting exons and SNPs, respectively. Genomic Primers designs primers around all exons of a selected gene, with adjustable flanking size (size between exon and primer). SNP Primers selects all the 'variations' in a GenBank file, and designs primers around those that are not marked 'ambiguous'. It also avoids designing primers overlapping nearby SNPs.
Lastly, cDNA primers designs primers around the open reading frames of cDNAs. The input is a cDNA list in GenBank format. When the ORF is the same size as the sequence, which is often the case with predicted genes, the program maximizes the product size. This allows batch design of primers to test for presence of absence of cDNAs in tissues.
PCR Suite was used for designing primers for mutation analysis in our Parkinson project (Science 2003 (5604): 256-9) and was proven very useful and time saving. It allowed us to quickly screen for presence of all cDNAs in the linkage region and find that one was missing: DJ1, the causative gene of Park7.
To optimise PCR efficacy, the program uses more stringent settings than the original PCR defaults, but the user can easily relax these and use the same program to design primers for those products that failed the first time.
PCR Suite is available at www.eur.nl/fgg/kgen/primer/.