High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS
| Autor: | Ivo Gut, Amar Gupta, Keith A. Bauer, Veeraiah Bodepudi, Thomas W. Myers, David H. Gelfand, Florence Mauger, Stephen Gordon Will |
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| Rok vydání: | 2012 |
| Předmět: |
animal structures
Genotype Genotyping Techniques Nitric Oxide Synthase Type I Alkalies 01 natural sciences Polymerase Chain Reaction Polymorphism Single Nucleotide 03 medical and health sciences chemistry.chemical_compound Primer dimer Genetics Humans Multiplex Genotyping Genetics (clinical) Polymerase Alleles 030304 developmental biology DNA Primers 0303 health sciences biology 010401 analytical chemistry Reproducibility of Results DNA Sequence Analysis DNA Hydrogen-Ion Concentration Ribonucleotides Molecular biology 0104 chemical sciences Diphosphates genomic DNA chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein RNA RNA Long Noncoding Primer (molecular biology) In silico PCR |
| Zdroj: | Human Mutation Human Mutation; Vol 34 |
| ISSN: | 1098-1004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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