title: Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.
abstract: The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm 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 degrees 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.
authors.affiliation: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, A-6020, Innsbruck, Austria. christian.huber@uibk.ac.at
authors.forename: C G
authors.initials: CG
authors.lastname: Huber
authors.forename: A
authors.initials: A
authors.lastname: Premstaller
authors.forename: W
authors.initials: W
authors.lastname: Xiao
authors.forename: H
authors.initials: H
authors.lastname: Oberacher
authors.forename: G K
authors.initials: GK
authors.lastname: Bonn
authors.forename: P J
authors.initials: PJ
authors.lastname: Oefner
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004247:DNA