Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis

Abstract

Real time PCR is the mainstay of current nucleic acid assays, underpinning applications in forensic science, point-of-care diagnostics and detection of bioterrorism agents. Despite its broad utility, the search for new tests continues, inspired by second and third generation DNA sequencing technologies and fuelled by progress in single molecule fluorescence spectroscopy, nanotechnology and microfabrication. These new methods promise the direct detection of nucleic acids without the need for enzymatic amplification. In this feature article, we provide a chemist's perspective on this multidisciplinary area, introducing the concepts of single molecule detection then focussing on the selection of labels and probe chemistry suitable for generating a signal detectable by ultrasensitive fluorescence spectroscopy. Finally, we discuss the further developments that are required to incorporate these detection platforms into integrated 'sample-in-answer-out' instruments, capable of detecting many target sequences in a matter of minutes.

Sandwich assay for mixed-sequence recognition of double-stranded DNA: Invader-based detection of targets specific to foodborne pathogens

“…1-6 For example, there is increased demand to replace culture-growing and PCR-based methods used for detection of microbial pathogens, with faster and less resource-intensive approaches. 4,7-9 Methods based on nucleic acid hybridization are of particular interest due to the predictability and specificity of Watson-Crick base pairing.…”

“…However, it is attractive to develop methods that detect dsDNA instead as this eliminates the need for denaturation prior to analysis and potentially allows for amplification-free detection of genomic DNA. 6 Unfortunately, conventional dsDNA targeting probe strategies, such as triplex forming oligonucleotides, 13 peptide nucleic acids (PNAs), 14 and minor groove binding polyamides, 15 require polypurine targets and/or display other limitations, which reduces the number of suitable dsDNA targets.…”

“…Importantly, this assay potentially eliminates the need for DNA denaturation prior to analysis. This, along with the possibility of coupling the assay with existing sample preparation and enrichment protocols, and integrating it with microarrays 4 and/or single molecule detection techniques, 6 suggest that exciting applications of Invader-based sandwich assays in nucleic acid diagnostics and biotechnology are possible. …”

Quantifying molecular colocalization in live cell fluorescence microscopy

“…The right image resembles a reconstruction of the left image, where areas of high colocalization can directly be classified. [1][2][3][4][5][6][7][8][9][10][11][12]. In FRET molecular interaction partners, such as proteins, antibodies, or short DNA or RNA strands, are labeled with donor and acceptor fluorophores that need to be in close proximity (1-10 nm) to each other to ensure the efficient transfer of excitation energy.…”

Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

“…Although Taqman probes35–38 or molecular beacons39, 40 combined with PCR have enabled separation‐free detection of DNA targets in solutions, incomplete quenching of free probes often leads to high fluorescence background and low signal‐to‐noise ratio 41. Alternatively, the advancement of single molecule spectroscopy (SMS) and single molecule probe strategies facilitate homogeneous, separation‐free detection with high sensitivity 42–52. As opposed to conventional ensemble detection methods that measure averaged fluorescence from the entire analyte population, SMS measures fluorescent bursts emitted from individual molecules as they pass through a femtoliter‐sized laser detection volume.…”