Trinucleotide rolling circle amplification: A novel method for the detection of rna and dna

Jean Marc Zingg, Sylvia Daunert

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number15
Pages (from-to)1-20
Number of pages20
JournalMethods and Protocols
Volume1
Issue number2
DOIs
StatePublished - 2018

Keywords

  • Affinity column
  • DNA
  • Detection
  • Diagnostic
  • Human papilloma virus
  • Isothermal amplification
  • Lateral flow assay
  • Noro virus
  • Point of care test
  • RNA
  • Rolling circle amplification
  • Usutu virus
  • Zika virus

ASJC Scopus subject areas

  • Biotechnology
  • Structural Biology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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