1 research outputs found
Chemical-Oxidation Cleavage Triggered Isothermal Exponential Amplification Reaction for Attomole Gene-Specific Methylation Analysis
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<sup>7</sup> 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 (<i>S</i>/<i>N</i> = 3) by fluorescence, and 576 aM (<i>S</i>/<i>N</i> = 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