Detection of Nucleic Acid Targets Using Ramified Rolling Circle DNA Amplification: A Single Nucleotide Polymorphism Assay Model

摘要

BackgroundIsothermal amplification methods provide alternatives to PCR that may be preferable for some nucleic acid target detection tasks. Among current isothermal target detection methods, ramified rolling circle amplification (RAM) of single-stranded DNA circles that are formed by ligation of linear DNA probes (C-probes or padlock probes) offers a unique target detection system by linked primers and a simple amplification system that is unconstrained by the target’s sequence context. Earlier implementations of RAM-based target detection were reported to be limited by background noise, due in part to unligated C-probe in the amplification reaction. We show here that a target-detection system using a biotinylated target-capture probe together with automated bead-handling reduces or eliminates background amplification noise. We demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA.