17-10035 CpG MethylQuest DNA Isolation Kit

17-10035
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      Overview

      Replacement Information
      Description
      Catalogue Number17-10035
      Trade Name
      • Chemicon
      • MethylQuest
      DescriptionCpG MethylQuest DNA Isolation Kit
      OverviewThe CpG MethylQuest DNA Isolation Kit allows for simple, effective and rapid enrichment of methylated DNA from genomic samples. Featuring a high affinity recombinant methyl-binding domain (MBD) protein pre-bound to magnetic beads, a rapid protocol, and a simple elution procedure that yields methylated DNA ready for downstream analysis, this high affinity kit enriches for methylated DNA from as little as 1 ng of DNA!

      CpG MethylQuest Advantages
      • Specific enrichment of methylated DNA fragments
      • No detectable binding of unmethylated or hemimethylated regions
      • Simple and fast 2-hour magnetic bead-based protocol
      • GST-MBD2b capture protein pre-bound to magnetic beads for consistent results
      • Reliable performance from 1 ng to 1 μg of DNA
      • Elute ready-to-use DNA & avoid additional cleanup steps that reduce yields
      Background InformationMethylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of many eukaryotic genes. In normal cells methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG-islands remain unmethylated. The exceptions are the extensive methylation of CpG islands associated with transcriptional inactivation of regulatory regions of imprinted genes and genes on the inactive X-chromosome of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers.

      To evalute the methylation status of either a specific locus or an entire genome, the isolation and enrichment of methylated DNA can be a useful first step. To enable this enrichement, the CpG MethylQuest DNA Isolation Kit utilizes a high-affinity GST-MBD protein pre-bound to a magnetic bead.

      This purified recombinant protein contains the methyl binding domain (MBD) of the mouse MBD2b protein fused to a glutathione-S-transferase protein (GST) from S. japonicum separated by a linker containing a thrombin cleavage site. The MBD from the MBD2b protein has the highest affinity among the known methyl CpG binding proteins for Me-CpG sites and the lowest cross reactivity with unmethylated CpG sequences (Fraga M.F., et al. (2003). Nuc. Acids Res, 31, 1765–1774). These properties allow for very specific enrichment of methylated regions.

      In addition to its high affinity for methylated DNA, the CpG MethylQuest protein binds to methylated CpG regardless of sequence context. This is in contrast to isolation of methylated DNA using proteins such as MeCP2, which requires a run of A-Ts near a CpG site. This combination of sequence independent, high affinity binding to methylated CpG sites and extremely low affinity for non-methylated sites results in a greater number of methylated CpG sites being recognized allowing for comprehensive analysis of methylation patterns across the genome.
      Materials Required but Not Delivered
      • Microcentrifuge tubes (~1.5 mL polypropylene)
      • Microcentrifuge
      • Pipettes and pipette tips (aerosol barrier tips are suggested to minimize potential cross contamination)
      • Sample mixing equipment (shaker or tube rotator)
      • Thermocycler or real-time PCR instrument
      • PCR reagents for control reactions (e.g. Novagen NovaTaq™ Hot Start DNA Polymerase cat# 71091-3 and 10 mM dNTP mix cat# 71004-3 or NovaTaq™ Hot Start Master Mix Kit cat# 71676-3)
      • DNAse-free ultrapure water (e.g. Millipore cat# 3098)
      • Rare-earth magnet or magnetic rack (e.g. Millipore Magna GrIP™ Rack cat# 20-400 or Millipore PureProteome™ Magnetic Stand cat# LSKMAGS08)

      References
      Product Information
      Components
      • CpG MethylQuest Binding Buffer
      • CpG MethylQuest Magnetic Beads with pre-bound recombinant MBD protein
      • CpG MethylQuest Wash Buffer 1
      • CpG MethylQuest Wash Buffer 2
      • TE Buffer
      • Control HeLa DNA 110 ng, MseI cut, 2ng/µl
      • Positive Control Primers 10 µM each
      • Negative Control Primers 20 µM each
      Applications
      ApplicationThe CpG MethylQuest DNA Isolation Kit allows for simple, effective & rapid enrichment of methylated DNA from genomic samples.
      Biological Information
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      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
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      Material Size30 Reactions
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