Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography.
Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.
Dickman
Mark J
MJ
Department of Chemical and Process Engineering, Biological and Environmental Systems, University of Sheffield, Sheffield S1 3JD, UK. mdickman@sheffield.ac.uk
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009696:Nucleic Acids / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D009696:Nucleic Acids