Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR.

ABSTRACT: A frequency-modulated fluorescence encoding method was used as a means to increase the number of fluorophores monitored during infrared-mediated polymerase chain reaction. Laser lines at 488 nm and 561 nm were modulated at 73 and 137 Hz, respectively, exciting fluorescence from the dsDNA intercalating dye, EvaGreen, and the temperature insensitive dye, ROX. Emission was collected in a color-blind manner using a single photomultiplier tube for detection and demodulated by frequency analysis. The resulting frequency domain signal resolved the contribution from the two fluorophores as well as the background from the IR lamp. The detection method was successfully used to measure amplification of DNA samples containing 10(4)-10(7) starting copies of template producing an amplification efficienc

SUBMITTER: Schrell AM 

PROVIDER: S-EPMC4013171 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature