Analysis and purification of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Sizing of DNA fragments is a routine analysis traditionally performed on ag arose or polyacrylamide gels. Electrophoretic analysis is labor-intensive w ith only limited potential for automation. Recovery of DNA fragments from g els is cumbersome. We present data on automated, size-based separation of D NA fragments by ion-pair reversed-phase high performance liquid chromatogra phy (IP RP HPLC) - DNA chromatography - on the WAVE(R) DNA Fragment Analysi s System with the DNASep(R) cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with t he technology. Length-dependent separation of dsDNA fragments is sequence i ndependent and retention times are highly reproducible. The resolving capab ilities of DNA chromatography are illustrated by the analysis of multiple D NA size markers. Resolved dsDNA fragments are easily collected and are suit able for downstream applications such as sequencing and cloning. DNA chroma tography under denaturing conditions with fluorescently labeled DNA fragmen ts offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reli able and automated alternative to gel electrophoresis for the analysis of D NA fragments. (C) 2000 Elsevier Science BN. All rights reserved.