Abstract
Purpose: :
Our lab is involved in array screening for mammalian retinal gene expression. Real time PCR (RT–PCR) is used on a portion of the resultant data to confirm our results. MultiPriDe (Multiple Gene Primer Design) was developed to address a need for accurate and rapid design of RT–PCR primers. MultiPriDe automates the process of primer design, starting with a GeneID identifier for appropriate genes and selecting usable primer pairs. This script greatly reduces the time required to develop RT–PCR primers, reduces user–introduced errors that frequently plague manual sequence management, and allows for batch processing of RT–PCR targets.
Methods: :
MultiPriDe consists of 300 lines of code; this perl script is designed to accept batches of GeneID numbers and retrieve gene sequence and splice site information from linked GenBank entries. For each gene in the batch it first builds a FASTA–formatted mRNA sequence and, in combination with splice site data, then formulates a primer3 query, directed at a locally installed version of that package. The primers designed by MultiPriDe are chosen to span splice sites, allowing the end user to address possible RT–PCR contamination by persistent genomic DNA in their samples. Moreover, MultiPriDe uses as its default parameters a specific set of conditions empirically determined to give higher quality RT–PCR primers than default conditions. MultiPriDe automates the process and uses a local computer's processing power; this avoids bandwidth and processing bottlenecks associated with heavy use of a public machine serving the primer3 utility. End user administration of MultiPriDe requires basic computer management skills.
Results: :
Initial use of MultiPriDe for RT–PCR primer development generated 2 primer pairs for each splice site of 270 target genes in 2 hours, roughly 75% of which proved to be immediately successful in gene specific RT–PCR analysis. Individual sequential querying of a public primer3 server to generate the equivalent set of primers is estimated to have taken ten to fifteen times longer.
Conclusions: :
As quantitative RT–PCR methods are ideal for the confirmation of data derived from microarray expression studies, a tool such as MultiPriDe automates and optimizes a critical step, efficient RT–PCR primer design.
Keywords: gene microarray • gene screening