scholarly journals rEXPAR: an isothermal amplification scheme that is robust to autocatalytic parasites

2019 ◽  
Author(s):  
Georg Urtel ◽  
Jean-Christophe Galas ◽  
André Estevez-Torres
Keyword(s):  
Programming Languages ◽  
Dna Polymerase ◽  
Reaction Network ◽  
Isothermal Amplification ◽  
Sequence Design ◽  
Molecular Programming ◽  
Nicking Enzyme

ABSTRACTIn the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR, as well as in related molecular programming languages, such as the PEN DNA toolbox. Here we show that the nicking enzyme Nb.BssSI allows to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network.

The influence of SYBR Green I on isothermal amplification with Bst exo DNA polymerase.

Biomics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 268-273
Author(s):  
A.R. Gilvanov ◽  
A.R. Sakhabutdinova ◽  
A.V. Chemeris ◽  
R.R. Garafutdinov
Keyword(s):  
Dna Polymerase ◽  
Sybr Green ◽  
Isothermal Amplification ◽  
Sybr Green I

scholarly journals Overview of DNA Self-Assembling: Progresses in Biomedical Applications

Pharmaceutics ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 268 ◽  
Author(s):  
Andreia Jorge ◽  
Ramon Eritja
Keyword(s):  
Programming Languages ◽  
Biomedical Applications ◽  
Great Promise ◽  
Dna Nanostructures ◽  
Modular Assembly ◽  
Molecular Programming ◽  
Self Assembling ◽  
Theranostic Agents ◽  
Assembly Principles

Molecular self-assembling is ubiquitous in nature providing structural and functional machinery for the cells. In recent decades, material science has been inspired by the nature’s assembly principles to create artificially higher-order structures customized with therapeutic and targeting molecules, organic and inorganic fluorescent probes that have opened new perspectives for biomedical applications. Among these novel man-made materials, DNA nanostructures hold great promise for the modular assembly of biocompatible molecules at the nanoscale of multiple shapes and sizes, designed via molecular programming languages. Herein, we summarize the recent advances made in the designing of DNA nanostructures with special emphasis on their application in biomedical research as imaging and diagnostic platforms, drug, gene, and protein vehicles, as well as theranostic agents that are meant to operate in-cell and in-vivo.

scholarly journals Data on multimerization efficiency for short linear DNA templates and phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase

Data in Brief ◽  
2020 ◽  
Vol 29 ◽  
pp. 105188 ◽  
Author(s):  
Ravil R. Garafutdinov ◽  
Assol R. Sakhabutdinova ◽  
Maxim S. Kupryushkin ◽  
Dmitrii V. Pyshnyi
Keyword(s):  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Dna Templates ◽  
Linear Dna

scholarly journals Enzyme-free nucleic acid dynamical systems

2017 ◽  
Author(s):  
Niranjan Srinivas ◽  
James Parkin ◽  
Georg Seelig ◽  
Erik Winfree ◽  
David Soloveichik
Keyword(s):  
Dynamical Systems ◽  
Programming Languages ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Complex Dynamics ◽  
Molecular Systems ◽  
Synthetic Dna ◽  
Molecular Programming ◽  
And Control ◽  
Dna Components

Chemistries exhibiting complex dynamics—from inorganic oscillators to gene regulatory networks—have been long known but either cannot be reprogrammed at will, or rely on the sophisticated chemistry underlying the central dogma. Can simpler molecular mechanisms, designed from scratch, exhibit the same range of behaviors? Abstract coupled chemical reactions have been proposed as a programming language for complex dynamics, along with their systematic implementation using short synthetic DNA molecules. We developed this technology for dynamical systems, identifying critical design principles and codifying them into a compiler automating the design process. Using this approach, we built an oscillator containing only DNA components, establishing that Watson-Crick base pairing interactions alone suffice for arbitrarily complex dynamics. Our results argue that autonomous molecular systems that interact with and control their chemical environment can be designed via molecular programming languages.

scholarly journals Room-temperature stable loop-mediated isothermal amplification (LAMP) reagents to detect leptospiral DNA

2021 ◽  
Vol 15 (4) ◽  
pp. 183-189
Author(s):  
Pui-Yuei Lee ◽  
Yien-Ping Wong ◽  
Shuhaidah Othman ◽  
Hui-Yee Chee
Keyword(s):  
Dna Polymerase ◽  
Cold Storage ◽  
Room Temperature ◽  
Rat Kidney ◽  
Isothermal Amplification ◽  
Storage Requirement ◽  
Loop Mediated Isothermal Amplification ◽  
Modified Method ◽  
Resource Limited ◽  
The Stability

Abstract Background Loop-mediated isothermal amplification (LAMP) is one of the most promising tools for rapidly detecting Leptospira spp. However, LAMP is hampered by cold storage to maintain the enzymatic activity of Bst DNA polymerase. Objective To overcome the drawback of cold storage requirement for LAMP reagents we modified the reagents by adding sucrose as stabilizer. We then sought to determine the stability at room temperature of the premixed LAMP reagents containing sucrose. Method Premixed LAMP reagents with sucrose and without sucrose were prepared. The prepared mixtures were stored at room temperature for up to 60 days, and were subjected to LAMP reactions at various intervals using rat kidney samples to detect leptospiral DNA. Results The premixed LAMP reagents with sucrose remained stable for 45 days while sucrose-free premixed LAMP reagents showed no amplification from day 1 of storage at room temperature up to day 14. Conclusion The LAMP reagent system can be refined by using sucrose as stabilizer, thus allowing their storage at room temperature without the need for cold storage. The modified method enables greater feasibility of LAMP for field surveillance and epidemiology in resource-limited settings.

scholarly journals A Stoichiometric and Pseudo Kinetic Model of Loop Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Navjot Kaur ◽  
Nikhil Thota ◽  
Bhushan Toley
Keyword(s):  
Kinetic Model ◽  
Reaction Pathway ◽  
Reaction Network ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Theoretical Understanding ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Applications

<p>Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications. The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays. </p>

scholarly journals COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2

Diagnostics ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 399 ◽  
Author(s):  
Ameh James ◽  
John Alawneh
Keyword(s):  
Cost Effective ◽  
Turnaround Time ◽  
Isothermal Amplification ◽  
Resource Limited Settings ◽  
Laboratory Staff ◽  
Resource Limited ◽  
Community Transmission ◽  
Rapid Tests ◽  
Novel Coronavirus ◽  
Nicking Enzyme

The current coronavirus disease 2019 (COVID-19) pandemic is largely driven by community transmission, after 2019 novel Coronavirus (2019-nCoV or SARS-CoV-2) crosses the borders. To stop the spread, rapid testing is required at community clinics and hospitals. These rapid tests should be comparable with the standard PCR technology. Isothermal amplification technology provides an excellent alternative that is highly amenable to resource limited settings, where expertise and infrastructure to support PCR are not available. In this review, we provide a brief description of isothermal amplification technology, its potential and the gaps that need to be considered for SARS-CoV-2 detection. Among this emerging technology, loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA) and Nicking enzyme-assisted reaction (NEAR) technologies have been identified as potential platforms that could be implemented at community level, without samples referral to a centralized laboratory and prolonged turnaround time associated with the standard COVID-19 RT-PCR test. LAMP, for example, has recently been shown to be comparable with PCR and could be performed in less than 30 min by non-laboratory staff, without RNA extractions commonly associated with PCR. Interestingly, NEAR (ID NOW™ COVID-19 (Abbott, IL, USA) was able to detect the virus in 5 min. More so, isothermal platforms are cost effective and could easily be scaled up to resource limited settings. Diagnostics developers, scientific community and commercial companies could consider this alternative method to help stop the spread of COVID-19.

scholarly journals Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus

2021 ◽  
Author(s):  
Andrea Salazar ◽  
Francisco M. Ochoa-Corona ◽  
Jennifer D. Olson ◽  
Binoy Babu ◽  
Mathews Paret
Keyword(s):  
Dna Polymerase ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Capture Method ◽  
Visual Diagnosis ◽  
Rose Rosette ◽  
The Rose

AbstractThis study explores the development of Loop-mediated isothermal amplification of DNA (LAMP) for detection of rose rosette emaravirus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense single-stranded RNA Emaravirus and causal agent of the rose rosette disease (RRD). Transmission of RRV is by Phyllocoptes fructiphilus, an eriophyid mite. Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear similar to herbicide damage. Two sets of RRV gene sequences composed of twenty-two accessions of RRV-P3 (RNA 3) and another twenty-four from RRV-P4 (RNA 4) were analyzed and two sets of four LAMP primers were designed for broad-range detection of RRV isolates. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. LAMP reactions were optimized for Bst 2.0 DNA polymerase using the outer RRV-F3/RRV-B3 primers, and internal RRV-FIP/RRV-BIP primers. LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. LAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) using Bst 2.0 LAMP was 1pg/μL and 1 fg/μL with GspSSD LD quantitative LAMP. The LoD of pre-reaction hydroxy naphthol blue (HNB, 120 μM) for colorimetric (visual) reactions was 10 pg/μL and 0.1 pg/μL using SYBR green I (1:10 dilution) in colorimetric post-reactions. No cross-reactivity was detected in LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses (INSV, ArMV, MSpV, TSWV, ApMV, PNRSV, ToRSV, and TMV), and one virus taxonomically related to RRV (HPWMoV). RNA from healthy rose tissues and non-template controls (water) were included in all LAMP assays.

A novel exonuclease-assisted isothermal nucleic acid amplification with ultrahigh specificity mediated by full-length Bst DNA polymerase

2018 ◽  
Vol 54 (75) ◽  
pp. 10562-10565 ◽  
Author(s):  
Xin Ye ◽  
Yang Li ◽  
Lijuan Wang ◽  
Xueen Fang ◽  
Jilie Kong
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Full Length ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification

A novel exonuclease-assisted isothermal amplification to amplify and determine nucleic acids very sensitively and with ultrahigh specificity.

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