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.

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.

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.

Immobilization of oligonucleotide-functionalized magnetic nanobeads in DNA-coils studied by electron microscopy and atomic force microscopy

2011 ◽  
Vol 1355 ◽  
Author(s):  
Mattias Strömberg ◽  
Sultan Akhtar ◽  
Klas Gunnarsson ◽  
Camilla Russell ◽  
David Herthnek ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Surface Coverage ◽  
Rolling Circle Amplification ◽  
High Surface ◽  
Rolling Circle ◽  
Force Microscopy ◽  
Atomic Force ◽  
Immobilized Beads ◽  
Magnetic Nanobeads

ABSTRACTImmobilization of oligonucleotide-functionalized magnetic nanobeads by hybridization in DNA-coils formed by rolling circle amplification has been investigated using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The TEM results supported earlier made observations that small beads with low oligonucleotide surface coverage preferably immobilize in the interior of the DNA-coils and do not tend to link several DNA-coils together whereas large beads with high surface coverage to a larger extent connect several DNA-coils together to clusters of several DNA-coils with beads. AFM provided direct visualization of the DNA-coils as thread-like objects. DNA-coils with immobilized beads appeared as a collection of beads with thread-like features in their near vicinity.

scholarly journals A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

2004 ◽  
Vol 78 (10) ◽  
pp. 4993-4998 ◽  
Author(s):  
Annabel Rector ◽  
Ruth Tachezy ◽  
Marc Van Ranst
Keyword(s):  
Restriction Enzyme ◽  
Rolling Circle Amplification ◽  
Enzyme Analysis ◽  
Amplification Efficiency ◽  
Sequence Information ◽  
Restriction Enzyme Digestion ◽  
Hpv 16 ◽  
Circular Dna ◽  
Rolling Circle ◽  
Papillomavirus Dna

ABSTRACT The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

scholarly journals Extrachromosomal circular DNA: a new potential role in cancer progression

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tianyi Wang ◽  
Haijian Zhang ◽  
Youlang Zhou ◽  
Jiahai Shi
Keyword(s):  
Genomic Instability ◽  
Cancer Progression ◽  
Formation Process ◽  
Potential Role ◽  
Human Cancer ◽  
Extrachromosomal Dna ◽  
Biological Functions ◽  
Circular Dna ◽  
Dna Molecule ◽  
Process Characteristics

AbstractExtrachromosomal circular DNA (eccDNA) is considered a circular DNA molecule that exists widely in nature and is independent of conventional chromosomes. eccDNA can be divided into small polydispersed circular DNA (spcDNA), telomeric circles (t-circles), microDNA, and extrachromosomal DNA (ecDNA) according to its size and sequence. Multiple studies have shown that eccDNA is the product of genomic instability, has rich and important biological functions, and is involved in the occurrence of many diseases, including cancer. In this review, we focus on the discovery history, formation process, characteristics, and physiological functions of eccDNAs; the potential functions of various eccDNAs in human cancer; and the research methods employed to study eccDNA.

scholarly journals Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease

2018 ◽  
Vol 108 (7) ◽  
pp. 902-909 ◽  
Author(s):  
Luz Marcela Yepes ◽  
Elizabeth Cieniewicz ◽  
Björn Krenz ◽  
Heather McLane ◽  
Jeremy R. Thompson ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Systemic Infection ◽  
Causative Role ◽  
Coding Region ◽  
Protein Coding ◽  
Rolling Circle ◽  
Reverse Transcription Pcr ◽  
Leaf Chlorosis ◽  
The Family

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.

scholarly journals Blueberry fruit drop-associated virus: A New Member of the Family Caulimoviridae Isolated From Blueberry Exhibiting Fruit-Drop Symptoms

Plant Disease ◽  
2016 ◽  
Vol 100 (11) ◽  
pp. 2211-2214 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Robert R. Martin
Keyword(s):  
Rolling Circle Amplification ◽  
The United States ◽  
Washington State ◽  
Cloning And Sequencing ◽  
Rolling Circle ◽  
Reading Frame ◽  
Fruit Drop ◽  
Perfect Correlation ◽  
The Family ◽  
Single Field

This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants exhibiting fruit-drop symptoms and named Blueberry fruit drop-associated virus (BFDaV). Blueberry fruit drop disease was first detected in blueberry plants in British Columbia, Canada in the late 1990s, and in a single field in northern Washington state in the United States in 2012. Infected bushes abort nearly 100% of their fruit about three weeks prior to harvest, when the berries are about 3 to 5 mm in diameter. At harvest, the affected plants appear taller than healthy ones as there is no fruit weighing down the branches. The virus was amplified from diseased material using rolling circle amplification, followed by enzyme digestion, cloning, and sequencing. The full genome of BFDaV is 9,850 bp in length and contains a single open reading frame, encoding for a polyprotein, and a large noncoding region. Based on the genome size and organization and phylogenetics, BFDaV is proposed as a new and the largest member of family Caulimoviridae. Finally, in mapping part of a field with fruit-drop symptoms, there was a nearly perfect correlation between the presence of the virus and fruit-drop symptoms.

Bead-based padlock rolling circle amplification for single DNA molecule counting

2013 ◽  
Vol 437 (1) ◽  
pp. 43-45 ◽  
Author(s):  
Kae Sato ◽  
Reina Ishii ◽  
Naoki Sasaki ◽  
Kiichi Sato ◽  
Mats Nilsson
Keyword(s):  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Dna Molecule ◽  
Single Dna Molecule
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