Application Note
Molecular Beacon Probes: Micro-Volume Fluorescence Measurement Of HPLC Isolated Probes
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•Application Note: Molecular Beacon Probes: Micro-Volume Fluorescence Measurement Of HPLC Isolated Probes
By Salvatore A.E. Marras, Ph.D, Public Health Research Institute, Newark, NJ
Joel Hansen, Ph.D, NanoDrop Technologies, Inc., Wilmington, DE
The NanoDrop® ND-3300 Fluorospectrometer uses a patented "cuvette-less" sample retention system for routine fluorescent measurements with as little as one micoliter. The system uses surface tension to hold micro-volume samples in place, allowing for instant, ‘at-strength' readings without dilution. Cleaning involves simply wiping or blotting the instrument's optical surfaces with a common lab wipe. These capabilities, combined with the intuitive, user-friendly software, provides quick and accurate fluorescence measurements with minimal consumption of sample.
Molecular Beacons are single-stranded oligonucleotide hybridization probes in a stem-and-loop configuration. The loop contains a probe sequence complementary to a target nucleic acid sequence while the stem is formed by annealing of complementary arm sequences located on either side of the probe sequence. A fluorophore (donor) is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm (Figure 1). Due to the close proximity of the donor and quencher when molecular beacon probes are free in solution, they transiently share electrons eliminating the donor's ability to fluoresce. In the presence of a target nucleic acid sequence, the probe hybridizes to the target and undergoes a conformational change, displacing the quencher and allowing the fluorophore to fluoresce brightly.
Non-hybridized molecular beacons are fluorescence-quenched and ideally suited to homogeneous assay design, making it unnecessary to isolate the probe-target hybrids to determine the presence of a nucleic acid target. In this study, conducted at the Public Health Research Institute (Newark, New Jersey), a series of molecular beacon probes were used to evaluate the ND-3300 Fluorospectrometer's performance against a reference Photon Technology International (PTI) QuantaMaster I spectrofluorometer.
Click Here To Download:•Application Note: Molecular Beacon Probes: Micro-Volume Fluorescence Measurement Of HPLC Isolated Probes
