Comparative quantification of nucleic acids using single-molecule detection and molecular beacons.
This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.
Zhang
Chun-Yang
CY
Mechanical Engineering Department & Whitaker Biomedical Engineering Institute, The Johns Hopkins University, Baltimore, MD 21218, USA.
Chao
Shu-Yi
SY
Wang
Tza-Huei
TH
D000818:Animals; D017403:In Situ Hybridization / Q000379:methods; D018613:Microscopy, Confocal; D036103:Nanotechnology; D009696:Nucleic Acids / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D009696:Nucleic Acids