OddCAPS: a simple, low-cost, universal technique for detecting single nucleotide variants

Derived cleaved amplified polymorphic sequence (dCAPS) assays are widely used to detect nucleotide variants, but suitable target sites are often limited because primer designs must preserve PCR amplification while creating restriction enzyme recognition sites. Here we report one-step dual-primer dCAPS PCR (OddCAPS), a modification that uses three primers in a single PCR amplification to overcome this constraint. An intermediate primer and a dCAPS primer sequentially introduce 1-2 base substitutions each, enabling up to four engineered base changes near the nucleotide of interest. By using the intermediate primer at 1/10-1/100 the concentration of the other primers, the desired product is generated directly in a single-tube, one-step PCR. Increasing the number of engineered substitutions improves the chance of using a researcher's preferred restriction enzyme. In principle, a set of eight common restriction enzymes is sufficient to detect any single-nucleotide variant in any biological or synthetic DNA sequence with this approach.