title: Chemical-oxidation cleavage triggered isothermal exponential amplification reaction for attomole gene-specific methylation analysis.
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.
authors: School of Chemistry and Chemical Engineering, Sun Yat-Sen University , Guangzhou, 510275 Guangdong, PR China.
authors: Yuzhi
authors: Y
authors: Xu
authors: Chang
authors: C
authors: Niu
authors: Xiaofen
authors: X
authors: Xiao
authors: Wenyuan
authors: W
authors: Zhu
authors: Zong
authors: Z
authors: Dai
authors: Xiaoyong
authors: X
authors: Zou
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D053837:DNA Cleavage; D019175:DNA Methylation; D004563:Electrochemistry; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D057230:Limit of Detection; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry*; D010084:Oxidation-Reduction
chemical_list: D009930:Organic Chemicals; D004247:DNA