scholarly journals Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

2002 ◽  
Vol 30 (14) ◽  
pp. 66e-66 ◽  
Author(s):  
M. Nilsson
Keyword(s):  
Real Time ◽  
Molecular Beacon ◽  
Rolling Circle Amplification ◽  
Real Time Monitoring ◽  
Rolling Circle ◽  
Modified Molecular Beacon

Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

Nano LIFE ◽  
2015 ◽  
Vol 05 (02) ◽  
pp. 1541002 ◽  
Author(s):  
Emil L. Kristoffersen ◽  
Maria Gonzalez ◽  
Magnus Stougaard ◽  
Cinzia Tesauro
Keyword(s):  
Real Time ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Drug Response ◽  
Molecular Beacon ◽  
Rolling Circle Amplification ◽  
Dna Topoisomerase ◽  
Rolling Circle ◽  
Topoisomerase Ib ◽  
Highly Sensitive

Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DNA-modifying enzymes like type IB Topoisomerases and subsequently amplified by a rolling circle amplification (RCA) mechanism. The RCA process can be followed in real-time by the addition of a molecular beacon with a fluorophore/quencher pair. Upon hybridization to the amplified product, the fluorophore/quencher pair is separated, giving rise to a fluorescent signal, measurable in pseudo real-time using a qPCR machine or in a fluorimeter. The RCA products in complex with the molecular beacon can subsequently be moved to microscopic slides and analyzed in a fluorescence microscope. We describe the proof of the principle of this molecular beacon-based method combining the qPCR readout format with the standard Rolling circle Enhanced Enzymatic Assay previously reported. Although the qPCR setup is less sensitive, it allows easy, fast, and high-throughput measurement of enzyme activities. Human Topoisomerase IB (TopIB) is a well-known target for the clinically used anticancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular TopIB activity affecting reversibly the Topoisomerase/DNA cleavage complexes. Therefore, we envisioned that the presented method may find use for the prediction of cellular drug response and for drug screening to discover novel molecules that specifically inhibit TopIB or other DNA-modifying enzymes.

scholarly journals Real-time Monitoring and Detection of Primer Generation-Rolling Circle Amplification of DNA Using an Ethidium Ion-selective Electrode

2016 ◽  
Vol 32 (5) ◽  
pp. 505-510 ◽  
Author(s):  
Ayaka SEICHI ◽  
Nanami KOZUKA ◽  
Yuko KASHIMA ◽  
Miyuki TABATA ◽  
Tatsuro GODA ◽  
...  
Keyword(s):  
Real Time ◽  
Rolling Circle Amplification ◽  
Ion Selective Electrode ◽  
Real Time Monitoring ◽  
Selective Electrode ◽  
Rolling Circle

Real-time monitoring of rolling circle amplification using aggregation-induced emission: applications in biological detection

2015 ◽  
Vol 51 (92) ◽  
pp. 16518-16521 ◽  
Author(s):  
Hong-Xin Jiang ◽  
Meng-Yao Zhao ◽  
Chen-Di Niu ◽  
De-Ming Kong
Keyword(s):  
Real Time ◽  
Rolling Circle Amplification ◽  
Real Time Monitoring ◽  
Biological Detection ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Aggregation Induced Emission

Real-time monitoring of rolling circle amplification (RCA) was achieved by the super-aggregation of a tetraphenylethene dye QAPTE along single-stranded DNA products and consequent enhanced aggregation-induced emission, it can work for all RCA reactions.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

A linear DNA probe as an alternative to a molecular beacon for improving the sensitivity of a homogenous fluorescence biosensing platform for DNA detection using target-primed rolling circle amplification

RSC Advances ◽  
2015 ◽  
Vol 5 (6) ◽  
pp. 4019-4025 ◽  
Author(s):  
Fulin Zhou ◽  
Baoxin Li ◽  
Jiyuan Ma
Keyword(s):  
Fluorescence Quenching ◽  
Molecular Beacon ◽  
Dna Detection ◽  
Rolling Circle Amplification ◽  
Dna Probes ◽  
Dna Probe ◽  
Rolling Circle ◽  
Hairpin Probe ◽  
Linear Dna ◽  
Signal Probe

Linear single-labeled DNA probes are used in this RCA-based fluorescence strategy for DNA detection, which could effectively avoid the fluorescence quenching between neighboring signal probes using hairpin probe as signal probe.

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