Discrimination among rice varieties based on rapid detection of single nucleotide polymorphisms by a newly developed method, mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) analysis

摘要

The method described here discriminates among rice cultivars based on single nucleotide polymorphisms (SNPs). The method is rapid (less than 1 hour), and combines PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We obtained sequence data from genome databases, and identified SNPs that could be used to distinguish among rice cultivars after digestion with restriction enzymes or urasil-DNA glycosylase (UDG). A crude extract was prepared by vortexing rice grains in a tube, and this was used as the template for PCR without further purification of genomic DNA. The PCR primers were designed based on the sequence around the SNP. The PCR was performed with a short extension time (2 s) and the amplicons were treated with restriction enzymes or UDG. Single-stranded DNA (ssDNA) obtained by alkali denaturation was analyzed by MALDI-TOF MS. This method, which we have used previously to identify transgenic genes in plants, successfully discriminated among rice cultivars based on SNPs. We added an asymmetric PCR to obtain greater quantities of ssDNA and a fast PCR step to increase the speed of amplification.