Abstract
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 T m values estimated from nucleotide sequences using the DINAMelt web server, measured T m values, and hybridization conditions yielding allele-specific signals. The estimated T m values were comparable to the measured T m 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 T m 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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Acknowledgments
This work was supported in part by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) and GreenTechno Project of the Ministry of Agriculture, Forestry, and Fisheries.
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Communicated by C. Quiros.
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Supplementary Fig. 1. Relationship between estimated Tm values (Tm-E) and measured Tm values (Tm-M) of complementary oligonucleotides (a) and mismatching oligonucleotides (b). Error bars indicate SE. (JPEG 195 kb)
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Supplementary Fig. 2. Relationship between estimated Tm values (a), measured Tm values (b), and temperatures for non-specific signal detection with mismatching oligonucleotides. (JPEG 234 kb)
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Supplementary Fig. 3. Effect of exposure time to X-ray film on allele-specific signal detection. Exposure time to the X-ray film was set at 1, 2, 5, 10, 20, or 40 min. Competitive oligonucleotides were not used. Each cell shows the result of signal detection using 0.1 x, 0.2 x, 0.5 x or 1 x SSC at 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. White cells indicate no signal detection, gray cells represent allele-specific signal detection, and black cells mean non-specific signal detection. The experiment was repeated three times, and results obtained more than twice are shown (TIFF 213 kb)
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Supplementary Fig. 4. Effect of competitive hybridization on allele-specific signal detection. Probe oligonucleotides and their competitive oligonucleotides were mixed at 1:0 or 1:5. Film exposure time was 40 min. Each cell shows the result of signal detection using 0.1 x, 0.2 x, 0.5 x or 1 x SSC at 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. White cells indicate no signal detection, gray cells show allele-specific signal detection, and black cells exhibit non-specific signal detection. The experiment was repeated three times, and results obtained more than twice are shown. (TIFF 126 kb)
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Shiokai, S., Kitashiba, H. & Nishio, T. Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes. Plant Cell Rep 29, 829–834 (2010). https://doi.org/10.1007/s00299-010-0867-z
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DOI: https://doi.org/10.1007/s00299-010-0867-z