DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious. In the present study, a small leaf disk of rice, 1-2 mm in diameter, punched by a mini cork borer was found to be directly usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1-1.5 mm in diameter were better than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates, named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis.
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