Improved RecA-Assisted Fluorescence Assay for DNA Strand Exchange Reaction

Abstract

Using the Force The mention of dark quenching may conjure up images of light-saber battles between the forces of ruddy-faced Good and mellifluous-voiced Evil. In the molecular sciences, however, the scenario is usually far more benign and significantly more practical. The process of dark quenching, in which bringing two molecules together, a fluor and a quencher, results in the abrogation of a light signal, can be seen as being the flip-side of fluorescence resonance energy transfer (FRET). FRET assays, where the close proximity of two molecules creates fluorescence, have been used previously to monitor, in real-time, the process of homologous recombination in vitro. A challenge with this and other fluorescence assays is to obtain a sufficiently high signal over the background, an issue that commonly confounds many such experiments. Kaboev et al. on p. 736 of this issue therefore decided to go over to the dark side by using a dsDNA template carrying a FAM fluor as well as a DABCYL quencher molecule. The propinquity of the fluor and quencher mean that no signal is seen until recombination takes place, at which time one of the DNA strands is liberated and the inhibition is released, resulting in a fluorescent signal. Using this modification, as well as designing the probe with a triple repeat of the target sequence, the authors were able to demonstrate an impressive 5- to 10-fold improvement in the signal-to-background ratio.

Field Demonstration of a Multiplexed Point-of-Care Diagnostic Platform for Plant Pathogens

“…This improvement also reduced the assay time by half while maintaining assay performance (Figure S3, Supporting Information). As RPA uses recombinase enzymes to assist primers/target recognition, , we believe that the same mechanism likely promoted the specific hybridization of the SERS nanotags to the barcode sequences of amplicons. With the help of a portable Raman spectrometer, the improved assay was successfully performed in a garden adjacent to our laboratory to detect a single diseased sample ( B. cinerea infected tomato plants, Figure S4, Supporting Information) to demonstrate a potential field application.…”

BisPNA‐assisted Detection of Double‐stranded DNA via Electrochemical Impedance Spectroscopy

“…DNA is in the form of duplex, which enhances the difficulty in detecting it. To improve this situation, two non-denaturing DNA hybridization methods, RecA-assisted and PNA-assisted DNA hybridization, are commonly in use [8][9][10]. Among those methods, dsDNA needs to be separated into two ssDNA via denaturation prior to detect DNA that will increase the complexity of experiment operation.…”

“…Traditional methods for double-stranded DNA (dsDNA) detection, such as southern blot and polymerase chain reaction (PCR), are based upon the specific recognition of single-stranded DNA (ssDNA) sequence via Watson-Crick base pairing [4][5][6]. RecA-assisted DNA hybridization can provide sequence-universal recognition of duplex using hybridization probe [8]. The denaturing procedure usually needs strict treatment which prohibits to separate target dsDNA in complete form, and spontaneous reannealing of two original ssDNA will interfere the hybridization between the target DNA and capture probe [7].…”

“…The denaturing procedure usually needs strict treatment which prohibits to separate target dsDNA in complete form, and spontaneous reannealing of two original ssDNA will interfere the hybridization between the target DNA and capture probe [7]. To improve this situation, two non-denaturing DNA hybridization methods, RecA-assisted and PNA-assisted DNA hybridization, are commonly in use [8][9][10]. RecA-assisted DNA hybridization can provide sequence-universal recognition of duplex using hybridization probe [8].…”

“…To improve this situation, two non-denaturing DNA hybridization methods, RecA-assisted and PNA-assisted DNA hybridization, are commonly in use [8][9][10]. RecA-assisted DNA hybridization can provide sequence-universal recognition of duplex using hybridization probe [8]. However, the recognition of dsDNA by oligonucleotides is much lower fidelity when RecA-assisted DNA hybridization is compared with protein-free DNA hybridization [11].…”