title: 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.
authors.affiliation: CEA/Institut de Génomique/Centre National de Génotypage, Bâtiment G2, Evry Cedex, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Mauger
authors.forename: David H
authors.initials: DH
authors.lastname: Gelfand
authors.forename: Amar
authors.initials: A
authors.lastname: Gupta
authors.forename: Veeraiah
authors.initials: V
authors.lastname: Bodepudi
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Will
authors.forename: Keith
authors.initials: K
authors.lastname: Bauer
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Myers
authors.forename: Ivo G
authors.initials: IG
authors.lastname: Gut
mesh_terms: D000468:Alkalies / Q000737:chemistry; D000483:Alleles; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D052248:Nitric Oxide Synthase Type I / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics; D015203:Reproducibility of Results; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000468:Alkalies; D017931:DNA Primers; D011756:Diphosphates; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D012265:Ribonucleotides; C107241:diphosphoric acid; D012313:RNA; D004247:DNA; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I