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Chemical-oxidation cleavage triggered isothermal exponential amplification reaction for attomole gene-specific methylation analysis

Authors :
Xiaoyong Zou
Yuzhi Xu
Wenyuan Zhu
Zong Dai
Xiaofen Xiao
Chang Niu
Source :
Analytical chemistry. 87(5)
Publication Year :
2015

Abstract

Genomic 5-methylcytosine (5-mC) modification is known to extensively regulate gene expression. The sensitive and convenient analysis of gene-specific methylation is wishful but challenging due to the lack of means that can sensitively and sequence-selectively discriminate 5-mC from cytosine without the need for polymerase chain reaction. Here we report a chemical-oxidation cleavage triggered exponential amplification reaction (EXPAR) method named COEXPAR for gene-specific methylation analysis. EXPAR was proved to not only have rapid amplification kinetics under isothermal condition but also show excellent sequence-selectivity and linear-dependence on EXPAR trigger. Further initiation of EXPAR by chemical-cleavage of DNA at 5-mC, the COEXPAR showed high specificity for methylated and nonmethylated DNA, and ∼10(7) copies of triggers were replicated in 20 min, which were used to quantify the methylation level at the methylation loci. As a result, the gene-specific methylation level of a p53 gene fragment, as a target model, was analyzed in two linear ranges of 10 fM-1 pM and 1 pM-10 nM, and limits of detection of 411 aM (S/N = 3) by fluorescence, and 576 aM (S/N = 3) by electrochemistry. The method fulfilled the assay in an isothermal way in ∼5 h without the need for tedious sample preparation and accurate thermocycling equipment, which is likely to be a facile and ultrasensitive way for gene-specific methylation analysis.

Details

ISSN :
15206882
Volume :
87
Issue :
5
Database :
OpenAIRE
Journal :
Analytical chemistry
Accession number :
edsair.doi.dedup.....ca56289e78981ca3af80b65467ee6798