Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 μm i.d. monolithic poly(styrene–divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is ba...

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Veröffentlicht in:Journal of biochemical and biophysical methods 2001-01, Vol.47 (1), p.5-19
Hauptverfasser: Huber, Christian G., Premstaller, Andreas, Xiao, Wen, Oberacher, Herbert, Bonn, Günther K., Oefner, Peter J.
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container_end_page 19
container_issue 1
container_start_page 5
container_title Journal of biochemical and biophysical methods
container_volume 47
creator Huber, Christian G.
Premstaller, Andreas
Xiao, Wen
Oberacher, Herbert
Bonn, Günther K.
Oefner, Peter J.
description The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 μm i.d. monolithic poly(styrene–divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75°C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.
doi_str_mv 10.1016/S0165-022X(00)00147-0
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source MEDLINE; Alma/SFX Local Collection
subjects Alleles
Base Sequence
Chromatography, High Pressure Liquid - instrumentation
Chromatography, High Pressure Liquid - methods
Denaturing high-performance liquid chromatography
DNA - genetics
DNA - isolation & purification
DNA Mutational Analysis - instrumentation
DNA Mutational Analysis - methods
DNA Primers - genetics
Heterozygote
Homozygote
Humans
Molecular Sequence Data
Monoliths
Mutation detection
Nucleic Acid Denaturation
Polymerase Chain Reaction
title Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns
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