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Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.

ABSTRACT: A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, 'primers' are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG-RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of 'primers' are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (approximately 60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG-RCA to various molecular diagnostic assays.

SUBMITTER: Murakami T

PROVIDER: S-EPMC2647323 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.

Murakami Taku T Sumaoka Jun J Komiyama Makoto M

Nucleic acids research 20081223 3

A simple isothermal nucleic-acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60 degrees C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous pr ...[more]

PMID: 19106144

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Project description:BACKGROUND: Isothermal 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. METHODOLOGY: Target 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. CONCLUSION: The 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.

Dataset Information

Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.

Publications

Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification.

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