Kawaguchi, K.; Komachiya, Y.; Muto, M.; Teshima, R.; Sakai, N.; Ohno, H.

Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assays have been widely performed historically to detect known base substitutions in many model organisms--notably Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Schizosaccharomyces pombe--where chemical mutagens that induce point mutations are frequently used. With the rise of whole-genome sequencing and genome editing technologies, dCAPS is increasingly applied to detect diverse nucleotide changes in additional organisms, including Drosophila, zebrafish, mammals, and agricultural crops (e.g., Oryza sativa and Hordeum vulgare). However, a key limitation of dCAPS is that genomic target sites amenable to primer designs that both preserve PCR amplification and create recognition sites for inexpensive, high-performance restriction enzymes are scarce. Here we report One-step dual-primer dCAPS (OddCAPS), a modification that uses three primers in one reaction to overcome this constraint. Two of these primers, an intermediate primer and a dCAPS primer, sequentially introduce 1-2 base substitutions each into the amplicon, 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, having eight common restriction enzymes (BamHI, EcoRI, NheI, SalI, BglII, ClaI, HindIII, and MluI) suffices to detect any single-nucleotide variant in any biological or synthetic DNA sequence with this approach.