Multiply Primed Rolling-Circle Amplification Method for the Amplification of Circular DNA Viruses

2010 ◽  
Vol 2010 (4) ◽  
pp. pdb.prot5415-pdb.prot5415 ◽  
Author(s):  
H. Stevens ◽  
A. Rector ◽  
M. Van Ranst
Keyword(s):  
Rolling Circle Amplification ◽  
Dna Viruses ◽  
Circular Dna ◽  
Rolling Circle ◽  
Amplification Method

scholarly journals Composite Analysis of the Virome and Bacteriome of HIV/HPV Co-Infected Women Reveals Proxies for Immunodeficiency

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 422 ◽  
Author(s):  
Juliana Siqueira ◽  
Gislaine Curty ◽  
Deng Xutao ◽  
Cristina Hofer ◽  
Elizabeth Machado ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Hpv Infection ◽  
Cervical Lesions ◽  
Dna Viruses ◽  
Circular Dna ◽  
Rolling Circle ◽  
Cell Counts ◽  
Increased Risk ◽  
High Risk Hpv ◽  
Ngs Data

The human cervical microbiome is complex, and its role in health and disease has just begun to be elucidated. In this study, 57 cervical swab samples from 19 HIV/HPV co-infected women were analyzed for both virome and bacteriome composition. Virome analysis focused on circular DNA viruses through rolling circle amplification followed by next-generation sequencing (NGS). Data were assigned to virus families and genera, and HPV types were identified. NGS data of bacterial 16S from a subset of 24 samples were assigned to operational taxonomic units and classified according to vaginal microbiome community state types (CSTs). Four viral families were found: Papillomaviridae, Anelloviridae, Genomoviridae, and Herpesviridae. Papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with multiple (≥3) high-risk HPV infection. Anellovirus read abundance was negatively correlated with host CD4+ T-cell counts. The bacteriome revealed the presence of CST III and CST IV, and women with ≥1% frequency of genomovirus or herpesvirus reads displayed an increased risk of carrying CST IV. By characterizing the composition of the cervical circular DNA viruses and the bacteriome of HIV/HPV co-infected women, we identified putative interactions between these two microorganism communities and their associations with patients’ clinical characteristics, notably immunodeficiency status.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Rafaela S. Fontenele ◽  
Matias Köhler ◽  
Lucas C. Majure ◽  
Jesús A. Avalos-Calleros ◽  
Gerardo R. Argüello-Astorga ◽  
...  
Keyword(s):  
South America ◽  
Rolling Circle Replication ◽  
Dna Virus ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Circular Dna ◽  
Rolling Circle ◽  
Rep Protein ◽  
Molecular Features ◽  
Viral Components

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

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.

CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection

2020 ◽  
Vol 158 ◽  
pp. 105239
Author(s):  
Gong Zhang ◽  
Lei Zhang ◽  
Jingtao Tong ◽  
Xianxian Zhao ◽  
Jianlin Ren
Keyword(s):  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Amplification Method ◽  
Mirna Detection

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 T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection

2014 ◽  
Vol 50 (13) ◽  
pp. 1576-1578 ◽  
Author(s):  
Liang Cui ◽  
Zhi Zhu ◽  
Ninghang Lin ◽  
Huimin Zhang ◽  
Zhichao Guan ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Ultrasensitive Detection ◽  
Rolling Circle ◽  
Enzymatic Amplification ◽  
Amplification Method ◽  
Microrna Detection ◽  
Dual Amplification ◽  
Amplification Strategy ◽  
Excellent Selectivity ◽  
T7 Exonuclease

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA–CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.

Identification of Extrachromosomal Circular DNA in Hop via Rolling Circle Amplification

2016 ◽  
Vol 148 (2-3) ◽  
pp. 237-240 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
David H. Gent ◽  
Robert R. Martin
Keyword(s):  
Electron Microscopy ◽  
Sequence Analysis ◽  
Higher Plants ◽  
Rolling Circle Amplification ◽  
Small Region ◽  
Extrachromosomal Dna ◽  
Circular Dna ◽  
Rolling Circle ◽  
Dna Molecule ◽  
The Family

During a survey for new viruses affecting hop plants, a circular DNA molecule was identified via rolling circle amplification (RCA) and later characterized. A small region of the 5.7-kb long molecule aligned with a microsatellite region in the Humulus lupulus genome, and no coding sequence was identified. Sequence analysis and literature review suggest that the small DNA molecule is an extranuclear DNA element, specifically, an extrachromosomal circular DNA (eccDNA), and its presence was confirmed by electron microscopy. This work is the first report of eccDNAs in the family Cannabaceae. Additionally, this work highlights the advantages of using RCA to study extrachromosomal DNA in higher plants.

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