Real-Time Rolling Circle Amplification for Protein Detection

2007 ◽  
Vol 79 (9) ◽  
pp. 3320-3329 ◽  
Author(s):  
Litao Yang ◽  
Christine W. Fung ◽  
Eun Jeong Cho ◽  
Andrew D. Ellington
Keyword(s):  
Real Time ◽  
Rolling Circle Amplification ◽  
Protein Detection ◽  
Rolling Circle

Protein detection based on rolling circle amplification sensors

Luminescence ◽  
2021 ◽  
Author(s):  
Haixia Shi ◽  
Jingjie Cui ◽  
Husseini Sulemana ◽  
Wunian Wang ◽  
Li Gao
Keyword(s):  
Rolling Circle Amplification ◽  
Protein Detection ◽  
Rolling Circle

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.

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.

An On-Nanoparticle Rolling-Circle Amplification Platform for Ultrasensitive Protein Detection in Biological Fluids

Small ◽  
2010 ◽  
Vol 6 (22) ◽  
pp. 2520-2525 ◽  
Author(s):  
Juan Yan ◽  
Shiping Song ◽  
Bing Li ◽  
Qingzhi Zhang ◽  
Qing Huang ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Rolling Circle Amplification ◽  
Protein Detection ◽  
Rolling Circle

scholarly journals Real-Time QCM Measurements of Rolling Circle Amplification Products

Proceedings ◽  
2017 ◽  
Vol 1 (4) ◽  
pp. 509 ◽  
Author(s):  
Narayanan Madaboosi ◽  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Jeanpierre Salas ◽  
Vasile Mecea ◽  
...  
Keyword(s):  
Real Time ◽  
Rolling Circle Amplification ◽  
Rolling Circle

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.

Sign in / Sign up

Export Citation Format

Share Document