Development of a routine procedure for single nucleotide polymorphism marker design based on the Tm-shift genotyping method

Abstract

To establish a routine procedure for SNP marker development, the previously reported Tm-shift SNP genotyping procedure was validated and extensively improved. The effect of introducing mismatched nucleotides into allele-specific primers, using alternative fluorescent dyes, and varying the DNA polymerase species on SNP discrimination was examined at more than 100 known SNP loci in a solanaceous crop, eggplant. As a result, it has been shown that a success rate over 80% can be achieved with Tm-shift SNP genotyping using directional trials with automatically designed primer sets and a routine protocol for the reaction condition tests.