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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
Author(s) -
Mauger Florence,
Gelfand David H.,
Gupta Amar,
Bodepudi Veeraiah,
Will Stephen G.,
Bauer Keith,
Myers Thomas W.,
Gut Ivo G.
Publication year - 2013
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22227
Subject(s) - biology , genotyping , microbiology and biotechnology , molecular inversion probe , multiplex polymerase chain reaction , multiplex , primer (cosmetics) , dna , polymerase chain reaction , primer dimer , in silico pcr , genomic dna , genetics , genotype , gene , chemistry , organic chemistry
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 .