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research article

Recombinase polymerase amplification in minimally buffered conditions

Tomar, Saurabh
•
Lavickova, Barbora
•
Guiducci, Carlotta
November 15, 2021
Biosensors and Bioelectronics

Application of recombinase polymerase amplification (RPA) for pH-based detection of DNA amplification has been investigated. Commercial RPA kits from TwistDx are modified to minimize their pH buffering capacity. Due to the RPA's unique biochemistry, removal of tris from the amplification kit is not enough to lower the buffering capacity of the RPA assay. Even in the absence of tris, RPA components in the commercial kit intrinsically buffer the pH. We show different strategies to minimize the buffering capacity of the RPA kit, while maintaining the amplification efficiency. Even in minimally buffered conditions, it is noticed that RPA's amplification yield is not high enough to overcome the assay's intrinsic buffering capacity. The effect of pyrophosphate precipitation in RPA on the reaction's pH have also been addressed. In conclusion, this work highlights strategies and considerations for the development of pH-based assays from nucleic acid amplification methods which involve ancillary enzymes that catalyze nucleotide hydrolysis.

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Type
research article
DOI
10.1016/j.bios.2021.113802
Author(s)
Tomar, Saurabh
Lavickova, Barbora
Guiducci, Carlotta
Date Issued

2021-11-15

Publisher

Elsevier

Published in
Biosensors and Bioelectronics
Volume

198

Article Number

113802

Subjects

Isothermal nucleic acid amplification

•

Recombinase Polymerase Amplification (RPA)

•

ISFET

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
CLSE  
Available on Infoscience
November 29, 2021
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/183272
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