Tri‐Nucleotide Rolling Circle amplification; a novel method for the detection of RNA and DNA

Most natural DNA and RNA are devoid of long Tri‐Nucleotide (TN) stretches of 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 in DNA or RNA is sequence‐specifically recognized, transferred, extended, amplified and detected by Padlock probes that consist entirely of nucleotides complementary to the nucleotides present in the target sequence (complementary TN‐information). Upon specific head‐to‐tail annealing and legation to the TN‐target sequence, these Padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by Tri‐Nucleotide Rolling Circle Amplification (TN‐RCA) into linear concatemeric long ssDNA. Since during TN‐RCA the MN (as dNTP) is not added to the reaction mixture during polymerization, only the correctly ligated circular Padlock will amplify, and background amplification will not occur with endogenous RNA or DNA (which mostly would require the presence of all fourd NTP). Therefore, in the absence of non‐specific amplification, various labeledd NTP can be added to the TN‐RCA reaction that enables the separation, isolation and detection of the amplified concatemeric linear ssDNA. Here we evaluate the TN‐RCA method with RNA/DNA derived from Zika virus and human papilloma virus(HPV). TN‐RCA is a novel isothermal amplification technique that can be used for rapid and sensitive sequence‐specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches (genomic DNA, bacteria, virus, mutations, polymorphisms, DNA methylation) with low background in short time. Support or Funding Information Coulter Foundation