High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products

Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.