fast magnetic assay for specific isolation of CpG-methylated DNA
Active Motif's MethylCollector™ Ultra offers a fast magnetic assay capable of efficiently isolating methylated CpG islands from fragmented genomic DNA*. MethylCollector Ultra is based on the Methylated CpG Island Recovery Assay (MIRA) which uses a MBD2b/MBD3L1 protein complex for improved enrichment of CpG dinucleotides. Enriched DNA can be used in many downstream applications, such as end point or real time PCR analysis of the methylation status of particular loci in normal and diseased samples, bisulfite conversion followed by cloning and sequencing, or amplification and labeling for microarray analysis.
To learn more about how the kit works, please select the Description tab below. To see data and results, including comparisons to alternative methods, select the Data tab below.
|MethylCollector™ Ultra||30 rxns||55005||$495||Buy Now|
|Fully Methylated Jurkat DNA||10 µg||55003||$250||Buy Now|
|Jurkat genomic DNA||10 µg||55007||$60||Buy Now|
|MethylCollector™ Ultra Manual|
|DNA Methylation Products Profile|
|MethylCollector & MethylCollector Ultra-Seq MOTIFvations June 13 Article|
|Fully Methylated Jurkat DNA|
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.
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.
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.
Figure 3: Real-time PCR analysis of MethylCollector™ Ultra using low salt (low stringency) conditions.
Figure 4: Methylation Analysis from as little as 1 ng (170 cells) DNA.
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.
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.
Click on image to enlarge size.
Figure 3: Next-Gen sequencing data generated using MethylCollector Ultra detects methylation at CpG shores.
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.