Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared Tm values estimated from nucleotide sequences using the DINAMelt web server, measured Tm values, and hybridization conditions yielding allele-specific signals. The estimated Tm values were comparable to the measured Tm values with small differences of less than 3°C for most of the probes. There were differences of approximately 14°C between the specific signal detection conditions and estimated Tm values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0× SSC corresponded to a difference of approximately 5°C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.
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