Enhanced discrimination of single nucleotide polymorphism in genotyping by phosphorothioate proofreading allele-specific amplification

There is a significant demand for sensitive, inexpensive, and flexible genotyping techniques that can be accomplished with reasonable throughput. Allele-specific amplification (ASA) has the advantage of combining the amplification and discrimination steps into a single reaction. However, mismatch amplification that occurs during traditional ASA limits its application for genotyping. Here, a modified ASA termed phosphorothioate proofreading allele-specific amplification (PP-ASA) is developed, for single nucleotide polymorphism (SNP) genotyping analysis. Using both 3′ end phosphorothioate modification of primers and DNA polymerase with proofreading activity completely eliminated mismatch amplifications, therefore enhancing discrimination between alleles for genotyping. The conditions for PP-ASA were optimized for template concentration and amplification cycle number as both were found to be critical for accurate genotyping. The utility of PP-ASA was assessed using both plasmid and genomic DNAs as templates and validated by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of 60 human DNA samples for two distinct SNPs. PP-ASA represents a reliable, flexible, and inexpensive assay for SNP genotyping; it could be integrated to chip- or PCR-array-based assays to improve the throughput and reduce the cost for SNP analyses.