Preparation of a Hyper‐cross‐linked Polymer Monolithic Column and Its Application to the Sensitive Determination of Genomic DNA Methylation |
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Authors: | Ming‐Luan Chen Yu‐Li Liu Xi‐Wen Xing Prof Xiang Zhou Prof Yu‐Qi Feng Prof Bi‐Feng Yuan |
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Institution: | Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072 (China), Fax: (+)?86‐27‐68755595 |
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Abstract: | A hyper‐cross‐linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium‐co‐vinylbenzene chloride‐co‐divinylbenzene) (MATE‐co‐VBC‐co‐DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE‐co‐VBC‐co‐DVB) monolithic column was demonstrated to have strong anion exchange/reversed‐phase (SAX/RP) mixed‐mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5‐methylcytosine (5‐mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise=3) of 5‐mC is 0.014 pmol and endogenous 5‐mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5‐azacytidine (5‐aza‐C) and 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) could induce a significant decrease of genome‐wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa). |
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Keywords: | liquid chromatography 5‐methylcytosine mixed‐mode retention nucleobases polymers |
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