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Issue 11, 2014
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Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR

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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 104–107 starting copies of template producing an amplification efficiency of 96%. The utility of this methodology was further demonstrated by simultaneous amplification of two genes from human genomic DNA using different color TaqMan probes. This method of multiplexing fluorescence detection with IR-qPCR is ideally suited as it allows isolation of the signals of interest from the background in the frequency domain and is expected to further reduce the complexity of multiplexed microfluidic IR-qPCR instrumentation.

Graphical abstract: Frequency-encoded laser-induced fluorescence for multiplexed detection in infrared-mediated quantitative PCR

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Article information


Submitted
17 Dec 2013
Accepted
11 Jan 2014
First published
13 Jan 2014

Analyst, 2014,139, 2695-2701
Article type
Paper
Author version available

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

A. M. Schrell and M. G. Roper, Analyst, 2014, 139, 2695
DOI: 10.1039/C3AN02334F

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