High Throughput Assay Design for Re-Sequencing of Exons
By Ellen Kloss
Despite the large number of candidate single nucleotide polymorphisms (SNPs) in public databases many have not been discovered, including some causing disease. In order to identify these variations it is necessary to conduct re-sequencing of large samples of patients and controls. We have optimized our design strategy to reduce experimental and in silico time and costs.
Our method of design selects the desired promoter and exon regions from genomic sequence and determines the minimum number of PCR products needed. Re-sequencing poses the challenge of amplifying and sequencing every exon. Our method starts with the most stringent parameters, then relaxes them in a hierarchically manner until primers are found for all exons. We then select one or both of the PCR primers to be used for sequencing, eliminating the need for design and purchase of additional sequencing primers (Vieux, et al. Biotech 32:S28). This method has worked for re-sequencing of human and mouse genes.