Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

Mok, Ellie; Wee, Eugene J. H.; Wang, Yuling; Trau, Matt

doi:10.1038/srep37837

Cited by 49 publications

(25 citation statements)

References 43 publications

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“…Hairpinshaped templates have been shown to improve the specificity over homologous sequences, although target-independent amplification is not delayed (22). Several additives such as graphene oxide (23), tetramethylammonium chloride (24), or single-strand binding protein (24,25) have been proposed to decrease these spurious reactions and increase assay sensitivity. Recently, Urtel et al (26) successfully suppressed background amplification using a template lacking one of the four nucleotides (dT-free template), a three-letter nicking site, and 2′-deoxyriboadenosine 5′-triphosphate (dATP)-free amplification mixture.…”

Section: Introductionmentioning

confidence: 99%

Isothermal digital detection of microRNAs using background-free molecular circuit

et al. 2020

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MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.

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Section: Introductionmentioning

confidence: 99%

Isothermal digital detection of microRNAs using background-free molecular circuit

et al. 2020

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“…It has been proven that trehalose is useful for the preservation of reagents (including primers, dNTPs and enzymes) for PCR 30–32 and LAMP 21 . Besides this application, it has been demonstrated to improve the analytical performance of other isothermal nucleic acid amplification methods, such as the exponential amplification reaction (EXPAR) 33 .…”

Section: Introductionmentioning

confidence: 99%

Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test

Diego

Fernández‐Soto

Crego-Vicente

et al. 2019

Sci Rep

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Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

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“…Non-specific amplification will increase the limit of detection and can decrease the experimental robustness. Optimizing solution conditions, adding ssDNA binding proteins and carbon sheets 39 , or adding other small molecules 40 can decrease non-specific amplification in EXPAR reactions, and would likely also be applicable in this reaction. Degradation of the reaction product was previously used to create a bistable switch from an EXPAR-type reaction 5 , and could be extended to suppress non-specific amplification or create threshold-based detection for targets above a chosen concentration.…”

Section: Resultsmentioning

confidence: 99%

First Characterization of a Biphasic, Switch-like DNA Amplification

Özay

Robertus

Negri

et al. 2017

Preprint

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We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.

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Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

Cited by 49 publications

References 43 publications

Isothermal digital detection of microRNAs using background-free molecular circuit

Isothermal digital detection of microRNAs using background-free molecular circuit

Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test

First Characterization of a Biphasic, Switch-like DNA Amplification

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