DNAメチル化解析キット MethylCollector™ Ultra

メチル化CpG DNAの濃縮

MethylCollector™ Ultra Kitは効率的にメチル化CpG DNAを限られた量の細胞や組織試料からメチル化CpG DNAを特異的に単離できるキットです。MethylCollector™ Ultra は複数の遺伝子座におけるメチル化状態のスクリーニングなど, さまざまなアプリケーションに使用することができ, がん組織や細胞の候補遺伝子のメチル化レベルを解析する際に極めて有用です。また通常の細胞分化や細胞老化などにおけるDNAメチル化の変化を検出する際にも使用できます。

 
Name Format Cat No. Price  
MethylCollector™ Ultra 30 rxns 55005 ¥94,000 Buy
Fully Methylated Jurkat DNA 10 µg 55003 ¥48,000 Buy
Jurkat genomic DNA 10 µg 55007 ¥16,000 Buy

MethylCollector Ultra Advantages

  • High affinity binding provides greater enrichment than other MBD capture or antibody-based methods
  • Fast magnetic bead based protocol is completed in less than 3 hours
  • Specifically detects methylated CpGs from 1 ng - 1 µg of DNA fragmented by sonication or enzymatic digestion
  • Includes positive control DNA and PCR primers to ensure success
  • Eluted DNA is suitable for various downstream applications

Methylated CpG Island Recovery Assay (MIRA)

MethylCollector™ Ultra is based on the Methylated CpG Island Recovery Assay (MIRA) which utilize a His-tagged recombinant methyl-binding protein complex, comprised of MBD2b/MBD3L1, that specifically binds methylated CpGs of genomic DNA fragments that have been prepared by sonication or enzymatic digestion. Nickel-coated magnetic beads capture the protein-DNA complexes which are separated from the rest of the genomic DNA using the included magnet. Optimized buffers ensure that fragments with little or no methylation are removed. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Following clean up, the eluted DNA is ready for use in PCR analysis or other downstream applications. See Flow Chart of MethylCollector™ Ultra process below.

The Methylated CpG Island Recovery Assay (MIRA) utilizes the high affinity of the MBD2b/MBD3L1 complex for methylated DNA1, to provide better enrichment than assays that use the methyl-binding protein MBD2 alone2.  MethylCollector™ Ultra can efficiently enrich for methylated DNA from as few as 170 cells (~1 ng DNA).

 

Flow Chart of the MethylCollector™ Ultra process.

 

References

1. Rauch, T. and Pfeifer, G. (2005) Lab. Investigations, 85:  1172-1180
2. Jiang, C.L. et al. (2004) J. Bio. Chem., 279:  52456-52464

Preparing DNA Samples for Next-Gen Sequencing following MethylCollector Ultra Enrichment

Enriched methylated DNA obtained using MethylCollector Ultra can be used directly in Next-Gen Sequencing sample processing protocols.

If you intend to perform Next-Gen sequencing analysis following MethylCollector Ultra enrichment of methylated DNA, please refer to the MethylCollector Ultra manual and the Illumina ChIP-Seq DNA Sample Prep Kit for instructions on how to prepare your DNA samples for Next-Gen sequencing.

Click on image to enlarge size.

Figure 1:  Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA correlates well with CpG density.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density except at CpG islands, which should be largely unmethylated.


Click on image to enlarge size.

Figure 2:  Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA is more sensitive and robust than data from other MBD enrichment kits.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit and a competitor’s MBD-based enrichment kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate whole-genome DNA methylation profiles. The top purple panel is data produced from MethylCollector Ultra and the orange panel is data from the Competitor M kit. MethylCollector Ultra identified more methylated regions and the peaks had greater intensity than peaks identified from the competitor kit.


Click on image to enlarge size.

Figure 3:  Next-Gen sequencing data generated using MethylCollector Ultra detects methylation at CpG shores.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows one of many examples of DNA methylation being detected at CpG shores rather than in the CpG island itself. This data agrees with recent findings showing that a large portion of methylated sites occur in these CpG shore regions that are adjacent to CpG islands.

Preparing DNA Samples for Next-Gen Sequencing following MethylCollector Ultra Enrichment

Enriched methylated DNA obtained using MethylCollector Ultra can be used directly in Next-Gen Sequencing sample processing protocols.

If you intend to perform Next-Gen sequencing analysis following MethylCollector Ultra enrichment of methylated DNA, please refer to the MethylCollector Ultra manual and the Illumina ChIP-Seq DNA Sample Prep Kit for instructions on how to prepare your DNA samples for Next-Gen sequencing.

Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA correlates with CpG density

Click on image to enlarge size.

Figure 1:  Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA correlates well with CpG density.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density except at CpG islands, which should be largely unmethylated.


Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA shows improved sensitivity

Click on image to enlarge size.

Figure 2:  Next-Gen sequencing data generated using MethylCollector Ultra enriched DNA is more sensitive and robust than data from other MBD enrichment kits.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit and a competitor’s MBD-based enrichment kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate whole-genome DNA methylation profiles. The top purple panel is data produced from MethylCollector Ultra and the orange panel is data from the Competitor M kit. MethylCollector Ultra identified more methylated regions and the peaks had greater intensity than peaks identified from the competitor kit.


Next-Gen sequencing data generated using MethylCollector Ultra detects methylation at CpG shores

Click on image to enlarge size.

Figure 3:  Next-Gen sequencing data generated using MethylCollector Ultra detects methylation at CpG shores.

DNA was enriched from 1 µg of human PBMC DNA using Active Motif’s MethylCollector Ultra Kit. Next-Gen sequencing was performed using the Illumina platform to generate 28 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows one of many examples of DNA methylation being detected at CpG shores rather than in the CpG island itself. This data agrees with recent findings showing that a large portion of methylated sites occur in these CpG shore regions that are adjacent to CpG islands.

MethylCollector™ Ultra Contents & Storage

His-MBD2b/MBD3L1 protein complex, High Salt Binding Buffer, Low Salt Binding Buffer, Elution Buffer AM1, Protease Inhibitors, Proteinase K, Proteinase K Stop Solution, Magnetic Nickel Beads, Mse I digested Human, male genomic DNA, NBR2 PCR Primer Mix methylated control, Xist PCR Primer Mix methylated control, GAPDH PCR Primer Mix unmethylated control, Glycogen, PCR Buffer, Loading Dye, Bar Magnet, Glue dots and PCR tubes. Storage conditions vary from room temperature to -20°C, please see manual for specific details. All products guaranteed for 6 months from date of receipt.

MethylCollector™ Ultra Publications

The following list represents selected publications in which Active Motif's assay has been used: