title: Ribo-polymerase chain reaction--a facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing.
abstract: We describe ribo-polymerase chain reaction (PCR), a method for the preparation of chimeric RNA/DNA. The RNA/DNA chimeric nucleic acids are generated directly from genomic DNA starting templates with two locus-specific primers, three nucleotides in their deoxy form and the fourth in its ribo form, a DNA polymerase capable of incorporating ribo bases, a suitable buffer, and thermal cycling. We have applied ribo-PCR to resequence DNA by directly fragmenting the RNA/DNA chimeras with alkali and analyzing the fragments by mass spectrometry (MS). Mass fingerprint is used to identify deviations from the reference sequence. This method readily detects homozygous sequence deviations as well as heterozygous positions directly from genomic DNA samples. With the high-throughput capability of MS, this facile method is well suited for screening DNA sequences of limited regions of the genome in a large number of individuals. It can also be used to sequence multiple distant genomic loci in a single reaction. This novel ribo-PCR resequencing protocol was applied to different genomic loci involving nitric oxide synthase 1 (NOS1) and H19 in 30 individuals and SLCO1B1 in 95 individuals.
authors.affiliation: CEA/Institut de Génomique/Centre National de Génotypage, Evry Cedex, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Mauger
authors.forename: Keith
authors.initials: K
authors.lastname: Bauer
authors.forename: Jérémy
authors.initials: J
authors.lastname: Semhoun
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Myers
authors.forename: David H
authors.initials: DH
authors.lastname: Gelfand
authors.forename: Ivo G
authors.initials: IG
authors.lastname: Gut
mesh_terms: D004247:DNA / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D027381:Liver-Specific Organic Anion Transporter 1; D013058:Mass Spectrometry; D052248:Nitric Oxide Synthase Type I / Q000235:genetics; D027361:Organic Anion Transporters / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C094335:H19 long non-coding RNA; D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C503999:SLCO1B1 protein, human; D012313:RNA; D004247:DNA; D052248:Nitric Oxide Synthase Type I