A robust method for detecting single-nucleotide changes as polymorphic markers by PCR

Numerous techniques in plant molecular genetic analysis, such as mapping and positional cloning techniques, rely on the availability of molecular markers that can differentiate between alleles at a particular locus. PCR-based cleaved amplified polymorphic sequences (CAPS) markers have been widely used as a means of rapidly and reliably detecting a single-base change that creates a unique restriction site in one of a pair of alleles. However, the majority of single nucleotide changes do not create such sites and thus cannot be used to create CAPS markers. In this paper, a modification of the CAPS technique that allows detection of most single-nucleotide changes by utilizing mismatched PCR primers is described. The mis-matches in the PCR primers, in combination with the single-nucleotide change, create a unique restriction site in one of the alleles.