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Recombinase assisted loop-mediated isothermal DNA amplification

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Abstract

Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3′ end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 102 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.

Graphical abstract: Recombinase assisted loop-mediated isothermal DNA amplification

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Publication details

The article was received on 02 Sep 2019, accepted on 08 Nov 2019 and first published on 03 Dec 2019


Article type: Paper
DOI: 10.1039/C9AN01701A
Analyst, 2020, Advance Article

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    Recombinase assisted loop-mediated isothermal DNA amplification

    G. Chen, R. Chen, S. Ding, M. Li, J. Wang, J. Zou, F. Du, J. Dong, X. Cui, X. Huang, Y. Deng and Z. Tang, Analyst, 2020, Advance Article , DOI: 10.1039/C9AN01701A

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