Sensitive fiber optics-based system for real-time detection of PCR-amplified DNA using molecular beacons

摘要

Abstract: Molecular Beacon hairpin shaped fluorescent oligonucleotide probes are powerful tools for quantifying specific nucleic acid sequences. Stratagene is developing a sensitive system, using these probes, for detecting and quantifying initial template copy number of nucleic acid sequences in real time during PCR amplification. The system allows parallel multiple fluorophore detection for many applications including allele discrimination and quantitative gene expression analysis. This instrument, combined with Stratagene's Sentinel Molecular Beacon kits, provides an effective system for molecular biology research. We report here the design and utility of an instrument that combines the capabilities of a microplate fluorescence reader with a PCR thermocycler into a low cost real time detection system. The instrument integrates a multiple fluorophore parallel fiber optic excitation and emission detection system, a precision X-Y translation stage, and a high performance thermoelectric temperature cycler with a computer controlled data collection and analysis system. The system uses standard PCR tubes, tube strips, and 96 well plates as the sample format. The result is a low cost, reliable, and easy to use system with premium performance for nucleic acid quantification in real time. 10!