Figure 1

Allele-specific amplification and detection of the PCR products. (A) Schematic representation of the allele-specific primer PCR method. 'Koshihikari' (Ksh) allele specific primer forms a perfect match at the 3' end (SNP) with Ksh DNA sequence (1) but forms a mismatch with Kal DNA (2). 'Kasalath' (Kal) allele specific primer similarly forms a 3' end match with Kal DNA (4) and 3' end mismatch with Ksh DNA (3). Both allele specific primer has an artificial mismatch at third base from 3' end (blue circle) according to the result from Table 1. (B) Allele-specific amplification of SNP marker S0285 detected EtBr after gel electrophoresis. Fluorescence of same samples were detected with a UV transilluminator at room temperature (25°C) or immediately after heating to 80°C using SYBR Green I. Ksh allele-specific primers (lane 1 and 2) and Kal allele-specific primers (lane 3 and 4) were used for PCR of Ksh genomic DNA (lane 1 and 3) and Kal genomic DNA (lane 2 and 4). The specificity of the reaction is evident. (C) The effect of temperature on SYBR Green I fluorescence. The green line indicates the fluorescence intensity of the sample in which amplification had occurred, and the blue line shows the amplification-independent background fluorescence. The ratio of both (signal/noise ratio) is shown in red. (D) The relationship between the number of SNPs and samples in a single PCR operation using 96-well plate. In a single PCR operation, 48 samples SNP genotype could be examined (multi-sample, Fig. 2), or 48 locus SNPs of one sample could be examined (multi-locus, Fig. 3)