High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

ABSTRACT

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 SNPs 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 NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.