MersV1, Main, Exploration, bibRecord, 001D89

Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

Identifieur interne : 001D89 ( Main/Exploration ); précédent : 001D88; suivant : 001D90

Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

Auteurs : Nidhi Sharma [États-Unis] ; Shuichi Hoshika [États-Unis] ; Daniel Hutter [États-Unis] ; Kevin M. Bradley [États-Unis] ; Steven A. Benner [États-Unis]

Source :

ChemBioChem [ 1439-4227 ] ; 2014-10-13.

RBID : ISTEX:0A8797721AA439305C2280653A3989814D48D564

Abstract

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single‐stranded primers into the duplex DNA product; these are then extended using a strand‐displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base‐pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self‐avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS‐RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

Unlike standard DNA primers that have a large background signal in isothermal amplification reactions, self‐avoiding molecular recognition system (SAMRS) components are modified nucleotides that eliminate these effects when added to primers, which allows for a real‐time fluorescence output. An assay to detect MERS RNA using SAMRS primers was optimized for quick, isothermal amplification.

Url:

DOI: 10.1002/cbic.201402250

Affiliations:

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<front><div type="abstract" xml:lang="en">Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single‐stranded primers into the duplex DNA product; these are then extended using a strand‐displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base‐pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self‐avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS‐RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.</div>
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Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

Recombinase‐Based Isothermal Amplification of Nucleic Acids with Self‐Avoiding Molecular Recognition Systems (SAMRS)

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