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Single copy-sensitive electrochemical assay for circulating methylated DNA in clinical samples with ultrahigh specificity based on a sequential discrimination-amplification strategy

Abstract

Tumor-related circulating methylated DNA represents only a small fraction of the total DNA in clinical samples (e.g. plasma), challenging the accurate analysis of specific DNA methylation patterns. Yet conventional assays based on real-time quantitative methylation-specific PCR (qMSP) are generally limited in detection sensitivity and specificity due to its non-specific amplification interference including primer dimer and off-target amplification. Here we propose a single copy-sensitive electrochemical assay for circulating methylated DNA with ultrahigh specificity on the basis of a sequential discrimination-amplification (SEDA) strategy. Methylated DNA rather than unmethylated one in bisulfite-modified sample is identified and amplified by asymmetric MSP to generate abundant biotin-labeled single-stranded amplicons with reduced primer-dimer artifacts. Self-assembled tetrahedral DNA probes, which are readily decorated on electrode surface as nanostructured probes with ordered orientation and well controlled spacing, enable the highly efficient hybridization of the specific single-stranded amplicons due to the greatly increased target accessibility and significantly decreased noise. The interfacial hybridization event is quantitatively translated into electrochemical signals utilizing an enzymatic amplification. The proposed assay integrates dual sequence discrimination processes and cascade signal amplification processes, achieving the identification of as few as single methylated DNA molecule in the presence of a 1,000-fold excess of unmethylated alleles. Furthermore, the excellent assay performance enables tumor related methylation detection of lung cancer patients in 200 microlitre of plasma samples. Results are well consistent with those of clinical diagnosis, whereas conventional qMSP failed to detect the corresponding methylation pattern of these clinically confirmed positive patients in such trace amount of samples.

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Publication details

The article was received on 07 Mar 2017, accepted on 15 May 2017 and first published on 18 May 2017


Article type: Edge Article
DOI: 10.1039/C7SC01035D
Citation: Chem. Sci., 2017, Accepted Manuscript
  • Open access: Creative Commons BY license
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    Single copy-sensitive electrochemical assay for circulating methylated DNA in clinical samples with ultrahigh specificity based on a sequential discrimination-amplification strategy

    X. Wang, F. Chen, D. Zhang, Y. Zhao, J. Wei, L. Wang, S. Song, C. Fan and Y. Zhao, Chem. Sci., 2017, Accepted Manuscript , DOI: 10.1039/C7SC01035D

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