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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Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR

Clinica Chimica Acta ◽

10.1016/j.cca.2011.06.031 ◽

2011 ◽

Vol 412 (21-22) ◽

pp. 1905-1911 ◽

Cited By ~ 27

Author(s):

Yanwei Zhong ◽

Jiaqi Han ◽

Zhengsheng Zou ◽

Shuhong Liu ◽

Bo Tang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Liver Tissue ◽

Rolling Circle Amplification ◽

Circular Dna ◽

Rolling Circle ◽

Covalently Closed Circular Dna ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification

Nucleic Acids Research ◽

10.1093/nar/gkaa017 ◽

2020 ◽

Vol 48 (5) ◽

pp. e30-e30 ◽

Cited By ~ 11

Author(s):

Bo Tian ◽

Gabriel Antonio S Minero ◽

Jeppe Fock ◽

Martin Dufva ◽

Mikkel Fougt Hansen

Keyword(s):

Nucleic Acid ◽

False Positive ◽

Rolling Circle Amplification ◽

Background Level ◽

Test Sample ◽

Negative Control ◽

Nucleic Acid Amplification ◽

Rolling Circle ◽

Nonspecific Amplification ◽

Negative Controls

Abstract False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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The analysis of proteins and small molecules based on sterically tunable nucleic acid hyperbranched rolling circle amplification

Biosensors and Bioelectronics ◽

10.1016/j.bios.2016.12.016 ◽

2017 ◽

Vol 91 ◽

pp. 136-142 ◽

Cited By ~ 16

Author(s):

Hai Shi ◽

Xiaoxia Mao ◽

Xiaoxia Chen ◽

Zihan Wang ◽

Keming Wang ◽

...

Keyword(s):

Nucleic Acid ◽

Small Molecules ◽

Rolling Circle Amplification ◽

Rolling Circle

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A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid

Analytica Chimica Acta ◽

10.1016/j.aca.2014.11.035 ◽

2015 ◽

Vol 856 ◽

pp. 103-109 ◽

Cited By ~ 20

Author(s):

Qingwang Xue ◽

Yanqin Lv ◽

Hui Cui ◽

Xiaohong Gu ◽

Shuqiu Zhang ◽

...

Keyword(s):

Nucleic Acid ◽

Rolling Circle Amplification ◽

Label Free ◽

Two Stage ◽

Exonuclease Iii ◽

Rolling Circle ◽

Dna Nanomachine

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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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