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

James H Smith,Thomas P Beals

doi:10.1371/journal.pone.0065053

Abstract

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.MethodologyTarget detection by RAM entails hybridization and ligation of a C-probe, followed by amplification and RAM signal detection. We evaluated RAM target detection in genomic DNA using recognition of a human Factor V gene single nucleotide polymorphism (G1691A) as a model. Locus-specific C-probes were annealed and ligated to genomic DNAs that represent the 3 possible genotypes at this locus, then ligated C-probes were amplified by real time RAM. The majority of the steps in the assay were performed with a magnetic bead-based chemistry on an automated platform. We show that the specificity of C-probe ligation permits accurate genotyping of this polymorphism. The assay as described here eliminates some of the background noise previously described for C-probe ligation, RAM amplification assays.ConclusionThe methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal RAM amplification provide a practical approach for detecting DNA targets in complex mixtures.

Highlights

Several isothermal amplification formats, as well as the polymerase-chain reaction (PCR), have been developed for detection of nucleic acid targets in complex nucleic acid mixtures [1]
The methods and results presented here show that a combination of C-probe detection, automated sample processing, and isothermal rolling circle amplification (RAM) amplification provide a practical approach for detecting DNA targets in complex mixtures
Commercially available isothermal methods include PCR-like reactions that depend upon multiple enzymes and other components to perform the denaturation/strand displacement function, rather than the thermal cycling of PCR; these include helicase dependent amplification (HAD) [2] and recombinase polymerase amplification (RPA) [3]

Summary

Introduction

Several isothermal amplification formats, as well as the polymerase-chain reaction (PCR), have been developed for detection of nucleic acid targets in complex nucleic acid mixtures [1]. Like traditional PCR, these methods require a target-specific primer pair. RAM reactions require a single stranded circular DNA, a single enzyme and a primer pair. Following target recognition and linear C-probe ligation, the circularized single-stranded DNA (ssDNA). 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 demonstrate the system’s performance by detection of a single-nucleotide polymorphism in human genomic DNA

Methods

Results

Conclusion