Optimal DNA Templates for Rolling Circle Amplification Revealed by In Vitro Selection

Abstract

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

Branched rolling circle amplification method for measuring serum circulating microRNA levels for early breast cancer detection

“…With the purpose of improving the sensitivity, specificity, and simplicity of c‐miRNA analysis, many novel techniques have been developed. Among them, rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic process that uses unique DNA or RNA polymerases to generate long single‐stranded DNA (ssDNA) or RNA (ssRNA) with tens to hundreds of tandem repeats by continuously adding nucleotides (nt) to a primer of the circular template . Unlike qRT‐PCR, which not only needs two‐step reactions (reverse transcription and polymerase chain reaction) but also requires a thermal cycler and thermostable DNA polymerases, RCA can be produced at an isothermal temperature from room temperature to 37°C by one‐step reaction .…”

Circular Nucleic Acids: Discovery, Functions and Applications

“…This method could also be coupled with paper microfluidic technology for the detection of both small and macromolecular molecules . Using an optimal RCA template evolved from in vitro selection, the detection sensitivity of a reduced graphene oxide‐based method can be significantly improved . In contrast with aptamer recognition‐induced primer immobilization or release, some researchers made use of aptamer‐target recognition to inhibit the functions of primers, and then impede RCA reaction, leading to a decrease in scanometric or fluorescent intensities .…”

DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy

“…Rolling circle amplification (RCA) represents a novel way to approach precision DNA nanostructure construction. RCA creates long stranded structures with repeating DNA sequences through the use of a circular template and DNA polymerase, which copies the template thousands of times . This allows for the precise templating of the RCA strand.…”