Single-Molecule Detection and Probe Strategies for Rapid and Ultrasensitive Genomic Detection

Abstract

This paper reviews the current state-of-the-art development of single-molecule detection (SMD)-based methods for ultrasensitive and specific analysis of genomic sequences. We first discuss several newly devised single fluorescent probe strategies that allow separation-free detection of low-abundance DNA sequences, such as quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) technology and dual-color fluorescence coincidence and colocalization analysis. Various schemes toward single DNA sizing and sequencing in solutions or on surfaces are also reviewed. In the end, we summarize the different microfluidic approaches developed for use with SMD to facilitate rapid, low-volume and quantitative analysis, such as electrokinetic and hydrodynamic single-molecule manipulation techniques.

An all-in-one microfluidic device for parallel DNA extraction and gene analysis

“…Northrup et al 1998;Erill et al 2003;Shin et al 2003;Cady et al 2005;Chen et al 2005;Dorfman et al 2005;deMello 2006;Lehmann et al 2006;Niu et al 2006;Ohashi et al 2007;Pipper et al 2007;Pipper et al 2008;Zhang et al 2009), cell/particle microfluidic manipulation (El-Ali et al 2006), biochemical sensing (Janasek et al 2006), biosample preparation (Wen et al 2008;Price et al 2009), optofluidics (Psaltis et al 2006), magnetic resonance imaging (Harel 2009) and mass spectroscopy (Grym et al 2006). The combination of microfluidic device with confocal optical spectroscopy has created an ideal platform for single molecule study (Wang et al 2005;Yeh et al 2005;Rane et al 2010).…”

Quantum dots and single molecule detection enable highly sensitive analysis of genetic biomarkers

“…> 1.2) and a confocal pinhole [2]. When measuring samples containing lowconcentration targets (< 1 nM), the detected fluorescence signal becomes digital because the molecular occupancy (the average number of molecules residing within the detection volume at any time) is smaller than unity.…”

“…This enables FRET with minimal direct acceptor excitation and donoracceptor crosstalk, thereby permitting the design of FRET molecular sensors with extremely low intrinsic fluorescence backgrounds necessary for detecting biomolecular targets at low abundance. On the other hand, there is an also increasing interest in using confocal single-molecule spectroscopy (SMD) for genomic detection [2]. The driving force not only comes from its ultrahigh sensitivity that allows detection of low-abundance nucleic acids without the need for amplification but also from its potential in achieving high-accuracy quantification of rare targets via single-molecule sorting.…”

Digital quantification of mutant DNA in cancer patients

“…These are based on the direct visualization of individual fluorescently labeled DNA molecules without the need for enzymatic amplification [20,21 ] So far only one of these approaches has been used for the enumeration of point mutations in solution [22]. The assay is based on the allele-specific ligation of fluorescence resonance energy transfer (FRET) probes generating molecular beacons upon successful ligation.…”