Molecular validation of pathogen‐reduction technologies using rolling‐circle amplification coupled with real‐time PCR for torquetenovirus DNA quantification

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.

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

10.26434/chemrxiv.14577975 ◽

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.

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Multilaboratory Evaluation of Real-Time PCR Tests for Hepatitis B Virus DNA Quantification

Journal of Clinical Microbiology ◽

10.1128/jcm.00471-11 ◽

2011 ◽

Vol 49 (8) ◽

pp. 2854-2858 ◽

Cited By ~ 38

Author(s):

A. M. Caliendo ◽

A. Valsamakis ◽

J. W. Bremer ◽

A. Ferreira-Gonzalez ◽

S. Granger ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Real Time ◽

Real Time Pcr ◽

Dna Quantification ◽

Hepatitis B Virus Dna ◽

B Virus

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

Nano LIFE ◽

10.1142/s1793984415410020 ◽

2015 ◽

Vol 05 (02) ◽

pp. 1541002 ◽

Cited By ~ 1

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.

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