Rolling Circle Amplification Combined with Gold Nanoparticle Aggregates for Highly Sensitive Identification of Single-Nucleotide Polymorphisms

Citations Over TimeTop 10% of 2010 papers

Abstract

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay method for single-nucleotide polymorphism genotyping has been developed. A circular template is generated by ligation upon the recognition of a point mutation on DNA targets. An RCA amplification is then initiated using the circular template in the presence of Phi29 polymerase. The RCA product can be digested by a restricting endonuclease, and the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. This causes a colorimetric change of the solution as the indicator of the mutation occurrence, which can be detected using UV-vis spectroscopy or viewed by naked eyes. On the basis of the high amplification efficiency of Phi29 polymerase, a mutated target of approximately 70 fM can be detected in this assay. In addition, the protection of the circle template using phosphorothioated nucleotides allows the digestion reaction to be performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant targets even when the ratio of the wild-type to the mutant is 10,000:1. The developed RCA-based colorimetric detection scheme was demonstrated for SNP typing of beta-thalassemia gene at position -28 in genomic DNA.

Related Papers

• → Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads(2011)102 cited
• → Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification(2011)22 cited
• → Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples(2022)4 cited
• → [Progress in rolling circle amplification in biological detection].(2019)3 cited
• → Improvement of rolling circle amplification efficiency with gold nanoparticles(2015)2 cited