Detection of purine and pyrimidine nucleotides and underivatized nucleic

Abstract

Sinusoidal voltammetry was employed to detect both purine and pyrimidine-based nucleic acids. Adenine and cytosine, representing these two classes of nucleic acids, could be detected with nanomolar detection limits at a copper electrode under these conditions, where the sensitivity for adenine was much higher than that for cytosine. Detection limits for purine-containing nucleotides (e.g., adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP)) were on the order of 70-200 nM using this method. These detection limits are achieved for native nucleotides and are over two orders of magnitude lower than those found with UV absorbance detection. Pyrimidine-based nucleotides could also be detected with high sensitivity due to the presence of a sugar backbone which is electroactive at the copper surface. This detector is not fouled by the nucleotides and can be used for the sensitive detection of analyses eluting continuously from a chromatography column or a electrophoresis capillary. Entire nucleic acid molecules can be readily detected. For example, both single stranded and double stranded DNA is detected with a detection limit in the picomolar concentration range (i.e., 10.sup.-12 moles/L). In the present invention, the signal from a double stranded DNA is roughly twice that arising from a single stranded DNA strand of the same length.