High‐Specificity Single‐Tube Multiplex Genotyping Using Ribo‐ PAP PCR , Tag Primers, Alkali Cleavage of RNA / DNA Chimeras and MALDI ‐ TOF MS

Here, we describe a high‐throughput, single‐tube, allele‐specific ribonucleotide analog pyrophosphorolysis‐activated polymerization (ribo‐ PAP ) PCR multiplex genotyping and resequencing method. An RNA / DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele‐selective 5′‐tagged primers, a reverse primer, one nucleotide in the ribo‐form (90–100%), the other nucleotides in the deoxy‐form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA / DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele‐selective primers have a 5′ repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo‐ PAP PCR . The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo‐ PAP PCR . Many different tags can be analyzed simultaneously. The assay can genotype several SNP s in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS 1 and H 19 in 30 individuals in simplex ribo‐ PAP PCR and at two SLCO 1 B 1 loci in 95 individuals in duplex ribo‐ PAP PCR .