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Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA

Department of Biochemistry and Molecular Biology, University of Miami, Miami, FL 33136-6129, USA
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Methods Protoc. 2018, 1(2), 15; https://doi.org/10.3390/mps1020015
Received: 13 February 2018 / Revised: 16 April 2018 / Accepted: 19 April 2018 / Published: 26 April 2018
Most natural DNA and RNA are devoid of long trinucleotide (TN) sequences that lack one specific nucleotide (missing nucleotide (MN)). Here we developed a novel method that is based on rolling circle amplification (RCA), in which the TN-information of short TN stretches is sequence-specifically recognized, transferred, extended, amplified and detected by padlock probes that consist entirely of nucleotides complementary to the three nucleotides present in the target sequence (complementary TN-information). Upon specific head-to-tail annealing and ligation to the TN-target sequence, these padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by trinucleotide rolling circle amplification (TN-RCA). Since during TN-RCA the MN (as dNTP) is not added, background amplification is minimized with endogenous RNA/DNA (which mostly would require all four dNTP). Therefore, various labelled dNTP can be added to the TN-RCA reaction that enables the separation, isolation and detection of the amplified single-stranded DNA (ssDNA). Here the TN-RCA method is exemplified with RNA/DNA from Zika virus and from human papilloma virus (HPV). TN-RCA is a novel isothermal amplification technique that can be used for sensitive sequence-specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches with low background in short time. View Full-Text
Keywords: isothermal amplification; rolling circle amplification; DNA; RNA; detection; lateral flow assay; affinity column; point of care test; diagnostic; Zika virus; Usutu virus; Noro virus; human papilloma virus isothermal amplification; rolling circle amplification; DNA; RNA; detection; lateral flow assay; affinity column; point of care test; diagnostic; Zika virus; Usutu virus; Noro virus; human papilloma virus
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Zingg, J.-M.; Daunert, S. Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA. Methods Protoc. 2018, 1, 15.

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Methods Protoc., EISSN 2409-9279, Published by MDPI AG
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