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


  • Communicated by Mats Nilsson

  • Contract grant sponsors: French Ministry of Education Research (Ministère Délégué à l'Enseignement et à la Recherche); European Community's Seventh Framework Program (FP7/2007–2013) (grant no. Health-f4–2008-201418—READNA).

Correspondence to: Ivo Gut, CEA/Institut de Génomique/Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France. E-mail:


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.