DNAメチル化解析キット　MethylCollector™ Ultra

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

The ability of MethylCollector™ to specifically enrich for methylated DNA over alternative methods such as MBD-Biotin capture, or antibody-based immunoprecipitation is shown in Figure 1. A direct comparison of eluted DNA using PCR primers for both unmethylated and methylated promoters reveals the ability of MethylCollector™ Ultra to specifically isolate CpG-methylated DNA. DNA eluted from the other methods showed little to no enrichment as both unmethylated and methylated promoters amplified at approximately the same number of PCR cycles.

Figure 1: MethylCollector™ Kits enrich methylated CpGs better than alternative methods.

Real time PCR analysis was performed on 100 ng of human, male genomic DNA that was Mse I digested and tested in the MethylCollector™ Ultra Kit, Competitor MM Kit and using the anti-5-methyl cytidine antibody immunoprecipitation method. Eluted DNA was analyzed using PCR primers for both methylated, SNRPN (red), and unmethylated, FOXD2 (blue), promoters. Only the MethylCollector™ Kits show a nice enrichment of methylated DNA as seen with the clear separation in amplification cycles. The enriched methylated DNA amplifies much earlier than unmethylated DNA.

Specificity of the MethylCollector™ Ultra Kit was also determined by comparing both the unbound and eluted fractions under both high and low salt conditions (Figures 2 & 3). Quality control testing was performed on 100 ng of Mse I digested human, male genomic DNA included in the kit. Enrichment of CpG-methylated DNA was confirmed using real-time PCR with the provided NBR2 (methylated promoter), Xist (methylated promoter) and GAPDH (unmethylated promoter) PCR primer mixes. The methylated NBR2 and Xist promoters gave robust signals and showed strong enrichment in the eluted fraction as compare to the unbound fraction. The unmethylated GAPDH promoter was only detected in the unbound fractions. These results verify the specificity of the MBD2b/MBD3L1 protein complex for methylated DNA. The level of enrichment may vary depending on the source of the DNA sample and the locus being evaluated.

GAPDH, glyceraldehyde-3-phosphate dehydrogenase, is important for metabolism. Because this gene is often constitutively expressed, it is considered to be an actively transcribed housekeeping gene containing an unmethylated promoter in healthy tissues. The region amplified by this primer pair is 69 base pairs and contains 7 CpGs.

Xist, X inactive specific transcript, is a methylated promoter in human, male genomic DNA, but is non-methylated in females. The region amplified by this primer pair is 178 base pairs and contains 8 CpGs.

NBR2, neighbor of BRCA1 gene 2 is located near the breast cancer gene BRCA1. Evidence indicates that NBR2 and BRCA1 share a bi-directional promoter. This region of the NBR2 gene is methylated promoter in healthy tissues. The region amplified by this primer pair is 103 base pairs and contains 7 CpGs.

Figure 2: Real-time PCR analysis of MethylCollector™ Ultra using high salt (high stringency) conditions.

A comparison of unbound and eluted fractions under high salt binding conditions shows the specificity of the MBD2b/MBD3L1 protein complex for CpG-methylated DNA. The methylated NBR2 promoter shows a 10-fold enrichment of methylated DNA in eluted fraction as compared to the unbound fraction, while the unmethylated GAPDH promoter was only detected in the unbound fraction.

Figure 3: Real-time PCR analysis of MethylCollector™ Ultra using low salt (low stringency) conditions.

A comparison of unbound and eluted fractions under low salt binding conditions shows the specificity of the MBD2b/MBD3L1 protein complex for CpG-methylated DNA. The methylated Xist promoter shows a 10-fold enrichment of methylated DNA in eluted fraction as compared to the unbound fraction, while the unmethylated GAPDH promoter was only detected in the unbound fraction.

Figure 4: Methylation Analysis from as little as 1 ng (170 cells) DNA.

The MethylCollector™ Ultra Kit has been shown to specifically detect CpG-methylated DNA from as few as 170 cells (~1 ng DNA). Varying amounts of Mse I digested human, male genomic DNA was tested in the MethylCollector™ Ultra Kit. The unbound and eluted fractions were collected and analyzed using real-time PCR with the included Xist PCR primer mix. Methylated DNA was recovered from as little as 500 pg, but enrichment of methylated DNA is seen with 1 ng or more of input material.

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.

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:

“Epigenetic stability, adaptability, and reversibility in human embryonic stem cells.” by Tompkins J.D. et al. (2012) Proc Natl Acad Sci USA, 109(31):  12544-12549.

“DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas.” by Lai A.Y. et al. (2010) J Exp Med., 207(9):  1939-1950.

“Overexpression of X-linked genes in T cells from women with lupus.” by Hewagama A. et al. (2013) J Autoimmun., 41:  60-71.

“BIM promoter directly targeted by EBNA3C in polycomb-mediated repression by EB.” by Paschos K. et al. (2012) Nucleic Acids Res., 40(15):  7233-7246.

“Disruption of Dnmt1/PCNA/UHRF1 interactions promotes tumorigenesis from human and mice glial cells.” by Hervouet E. et al. (2010) PLoS One, 5(6):  e11333.

“Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines.” by Sugawara H. et al. (2011) Epigenetics, 6(4):  508-515.

“Oncogenic human papillomavirus imposes an instructive pattern of DNA methylation changes which parallel the natural history of cervical HPV infection in young women.” by Leonard S.M. et al. (2012) Carcinogenesis, 33(7):  1286-1293.