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Sensitive, Specific Isolation of Methylated DNA
Sponsored,vendor-submitted protocol    Published in November 2009 (p.27) DOI: 10.2144/000113004

Abstract

MethylCollector™ provides an efficient method for isolating CpG-methylated DNA from limited amounts of cell or tissue samples. MethylCollector enables rapid screening of the methylation levels across multiple loci, making the method especially useful for analyzing the methylation status of tumor tissues or cells and detecting changes in DNA methylation throughout normal cellular processes. The kit uses a recombinant methyl-binding protein to capture DNA, rather than antibody-based immunoprecipitations, greatly improving assay sensitivity.

MethylCollector™ provides an efficient method for isolating CpG-methylated DNA from limited amounts of cell or tissue samples. MethylCollector enables rapid screening of the methylation levels across multiple loci, making the method especially useful for analyzing the methylation status of tumor tissues or cells and detecting changes in DNA methylation throughout normal cellular processes. The kit uses a recombinant methyl-binding protein to capture DNA, rather than antibody-based immunoprecipitations, greatly improving assay sensitivity.

Introduction

In the MethylCollector method, His-tagged recombinant MBD2b protein specifically binds CpG-methylated DNA fragments that have been prepared by enzymatic digestion or sonication. These protein-DNA complexes are captured with nickel-coated magnetic beads and subsequent wash steps are performed with a stringent high-salt buffer to remove fragments with little or no methylation. The methylated DNA is then eluted from the beads (Figure 1). Because the MBD2b-binding of methylated DNA is highly efficient, analysis of the methylation status of a specific DNA locus can be performed on DNA isolated from less than 800 cells (∼5 ng DNA).


Figure 1. Schematic of MethylCollector™ procedure. (Click to enlarge)


Optimized Method Yields Better Results

By utilizing the highly specific DNA-binding properties of MBD2b in MethylCollector, capture of methylated DNA is efficiently performed in a simple 1-h incubation. Magnetic beads facilitate the isolation of captured methylated DNA with fast and simple wash steps. The captured methylated DNA is eluted after a 30-min incubation, and the DNA is ready for analysis by PCR (Figure 2). The MethylCollector Kit has been carefully optimized to yield specific results with 5 ng to 1 µg of sample DNA.


Figure 2. Isolation of methylated DNA. (Click to enlarge)


Complete Kit Ensures Success

MethylCollector is a rapid and efficient method to compare levels of methylation at user-specified genomic loci in DNA from tissue or cell culture samples. For successful comparison of methylation levels, the DNAs to be tested should be purified and fragmented by the same methods, and an equal amount of each sample should be used in the MethylCollector procedure. MethylCollector includes both positive and negative control DNAs, control PCR primers, and all the necessary buffers, magnetic beads and magnet for 25 reactions. For additional DNA methylation experiments, Fully Methylated Jurkat DNA is available separately as a control. Please visit www.activemotif.com/methylcollector for more information Correspondence

Address correspondence to Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150, Carlsbad, CA 92008, USA. Tel.: (877)222-9543; [email protected], www.activemotif.com

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