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

The high resolving power of the chromatographic separation of single- and d ouble-stranded nucleic acids in 200 mum i.d. monolithic poly(styrene-diviny lbenzene) capillary columns was utilized for mutation screening in polymera se chain reaction amplified polymorphic loci. Recognition of mutations is b ased on the separation of homo- and heteroduplex species by ion-pair revers ed-phase high-performance liquid chromatography (IP-RP-HPLC) under partiall y denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide subst itutions and combinations thereof were confidently identified in 413 bp amp licons 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 d egreesC taking advantage of the ability of IP-RP-HPLC to resolve single-str anded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to e lectrospray ionization mass spectrometry and promise increased sample throu ghput by operating in arrays similar to those already used in capillary ele ctrophoresis. (C) 2001 Published by Elsevier Science B.V.