Nondenaturing HPLC is used for the sizedependent separation of dsDNA molecules, which depends on the length of the molecules, but not the base composition. After polymerase chain reactions (PCR), the amplified DNA fragments are directly injected into the DHPLC analysis system. The column temperature is maintained at 50°C and DNA molecules remain double-stranded. The concentration of the eluent (acetonitrile) is increased with time.
The shorter DNA fragments will be eluted and detected first, followed by the longer fragments. The eluted DNA fragments are detected by the ultraviolet (UV) detector and the chromatographic peaks representing the corresponding DNA fragments are demonstrated on the computer screen. Similar to conventional gel electrophoresis, nondenaturing HPLC can accurately determine the size of the amplicons. Unlabeled products can be used for analysis even with a UV detector if the injection volumes are large.
Use of unlabeled products reduces the cost of analysis. With small injection volumes, reliable quantification can also be achieved by adding dsDNA intercalation dye such as SYBR Green I and measuring the green fluorescence with a fluorescence detector. The dye is mixed with the DNA samples after elution from the column (postcolumn addition). The throughput of such analyses can further be increased by multiplex PCR in which several fragments of different sizes are amplified in the same tube.