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Development of a technique based on the affinity resins for evaluation of DNA methylation status in vertebrates

Development of a technique based on the affinity resins for evaluation of DNA methylation status... The methods for synthesis and application of resins based on the functional domains of Kaiso and CpG-binding protein (CGBP), which can bind methylated and unmethylated CpG-dinucleotides, respectively, are shown. Kaiso resin was obtained by the affinity interaction of glutathione-sepharose with a chimeric protein, which is expressed in Escherichia coli and contain glutathione S-transferase (GST) and zinc finger domain of methyl-DNA-binding Kaiso protein within the same translation frame. Kaiso resin, like MBD-domain based resin, has an ability to bind methylated DNA. Experiments with the short DNA fragments demonstrated that methylated DNA is eluted from the resin by 0.7 M KCl, whereas unmethylated DNA is washed out by 0.2–0.5 M KCl after binding. Quantitative PCR showed that the enrichment with methylated p16 promoter region and the absence of accumulation of γ-actin unmethylated promoter were observed due to the binding of genomic DNA, isolated from the colo 320 cell line (human colorectal adenocarcinoma), with the Kaiso resin. The CGBP resin based on the CxxC domain of CGBP protein binds to the sequences which contain unmethylated CpG-dinucleotides. Our experiments also showed no effect of MBD3L1 protein on MBD2-resin capacity of binding with methylated DNA. The obtained resins can be applied to study methylation status of both specific DNA sequences and the whole genome. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biochemistry and Microbiology Springer Journals

Development of a technique based on the affinity resins for evaluation of DNA methylation status in vertebrates

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References (30)

Publisher
Springer Journals
Copyright
Copyright © 2010 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Medical Microbiology ; Microbiology ; Biochemistry, general
ISSN
0003-6838
eISSN
1608-3024
DOI
10.1134/S0003683810070033
Publisher site
See Article on Publisher Site

Abstract

The methods for synthesis and application of resins based on the functional domains of Kaiso and CpG-binding protein (CGBP), which can bind methylated and unmethylated CpG-dinucleotides, respectively, are shown. Kaiso resin was obtained by the affinity interaction of glutathione-sepharose with a chimeric protein, which is expressed in Escherichia coli and contain glutathione S-transferase (GST) and zinc finger domain of methyl-DNA-binding Kaiso protein within the same translation frame. Kaiso resin, like MBD-domain based resin, has an ability to bind methylated DNA. Experiments with the short DNA fragments demonstrated that methylated DNA is eluted from the resin by 0.7 M KCl, whereas unmethylated DNA is washed out by 0.2–0.5 M KCl after binding. Quantitative PCR showed that the enrichment with methylated p16 promoter region and the absence of accumulation of γ-actin unmethylated promoter were observed due to the binding of genomic DNA, isolated from the colo 320 cell line (human colorectal adenocarcinoma), with the Kaiso resin. The CGBP resin based on the CxxC domain of CGBP protein binds to the sequences which contain unmethylated CpG-dinucleotides. Our experiments also showed no effect of MBD3L1 protein on MBD2-resin capacity of binding with methylated DNA. The obtained resins can be applied to study methylation status of both specific DNA sequences and the whole genome.

Journal

Applied Biochemistry and Microbiology – Springer Journals

Published: Nov 3, 2010

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