Rapid detection of maize DNA sequence variation.
The allele-specific polymerase chain reaction (ASPCR) has been used to determine the genotype of maize lines at two loci, wx and NPI288. The ASPCR method uses allele-specific oligonucleotide primers in PCR amplifications to amplify and discriminate simultaneously between polymorphic alleles. The success of this technique relies on the specific failure of PCR to amplify with primers that do not perfectly match the DNA sequence of one of the allelic variants. Amplification results were evaluated by dot-blot hybridization using an alkaline-phosphatase-coupled probe. The technique's speed, accuracy, sensitivity, and high throughput make it valuable for plant-breeding applications.