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Ribo‐polymerase chain reaction—A facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing
Author(s) -
Mauger Florence,
Bauer Keith,
Semhoun Jérémy,
Myers Thomas W.,
Gelfand David H.,
Gut Ivo G.
Publication year - 2012
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.22061
Subject(s) - biology , dna nanoball sequencing , genomic dna , dna , polymerase chain reaction , inverse polymerase chain reaction , microbiology and biotechnology , sequencing by ligation , multiple displacement amplification , dna sequencing , primer dimer , genetics , nucleic acid , rna , computational biology , genomic library , gene , multiplex polymerase chain reaction , dna extraction , base sequence
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. Hum Mutat 33:1010–1015, 2012. © 2012 Wiley Periodicals, Inc.