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.
Highlights
Rolling circle amplification (RCA) occurs naturally to replicate the genomes of certain transposons, bacterial plasmids and viruses having circular genomes [1]
When compared to RCA, the ends of the padlock probe oligonucleotides in the trinucleotide rolling circle amplification (TN-RCA) method target DNA or RNA stretches in which one or more nucleotides are missing, facilitating correct annealing and ligation at low temperature
The base complementary to the missing base in the target sequence is completely absent in the TN-RCA padlock probe, so that upon ligation, the circular template used for amplification consists only of three nucleotides
Summary
Rolling circle amplification (RCA) occurs naturally to replicate the genomes of certain transposons, bacterial plasmids and viruses having circular genomes [1]. Rolling circle amplification has been developed for a number of in vitro applications, such as for isothermal amplification of circularized DNA templates generating amplification products for sequencing, library construction, bioengineering or diagnosis [2,3,4]. Circularized DNA templates are formed upon connecting the ends of linear DNA by ligases such as T4 ligase from Bacteriophage T4 or Paramecium bursaria chlorella virus (PBCV-1) ligase from Chlorella virus. Circular DNA is formed by annealing head-to-tail single-stranded linear 5 -phosphorylated padlock probes of typically 50–200 bp to linear target DNA or RNA and ligase-mediated joining of their ends [5]. Whereas RCA is a linear amplification technique, a number of modifications in the RCA technique allow quasi-exponential amplification, such as the ramification or cascade amplification method (RAM), RCA coupled with loop-mediated amplification (RCA-LAMP), or as recently demonstrated by circle-to-circle amplification (C2CA) RCA [2,3,14,15,16,17,18]
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