Rapid amperometric verification of PCR amplification of DNA.

Abstract

Amplification of an 800-base template was verified in a 10-min test on a 2-microL sample of the PCR product solution. For verification, digoxigeninylated primers and biotinylated d-UTP-16-biotin were added to the amplification solution. The resulting amplified product was digoxigeninlabeled at its 3'-end and was also labeled with multiple biotin functions along its chain. The detecting electrode was coated with an electron-conducting redox hydrogel to which anti-digoxin monoclonal antibody was covalently bound. The amplified DNA was captured by the electrode through conjugation of its 3'-digoxigenin with the antibody. Exposure to a solution of horseradish peroxidase-labeled avidin led to capture of the enzyme and switched the redox hydrogel from a noncatalyst to catalyst for H2O2 electroreduction. The switching resulted in an H2O2 electroreduction current density of 2.1 +/- 0.9 microA cm-2 in 10-4 M H2O2 at Ag/AgCl potential and at 25 degrees C.