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Active Motif's MethylCollector™ product line offers fast magnetic assays capable of efficiently isolating methylated CpG islands from fragmented genomic DNA*. Enriched DNA can be used in many downstream applications, such as endpoint or realtime 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.
*Technology covered under U.S. Patent No. 7,425,415.
| Name | Format | Cat. No. | Price | |
|---|---|---|---|---|
| MethylCollector™ Ultra | 30 rxns | 55005 | $435 |
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| MethylCollector™ | 25 rxns | 55002 | $360 |
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| Fully Methylated Jurkat DNA | 10 µg | 55003 | $230 |
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| MethylCollector™ Manual |
| MethylCollector™ Ultra Manual |
| DNA Methylation Products Profile |
| Chromatin & Epigenetics Products Brochure |
| Fully Methylated Jurkat DNA |
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
Both MethylCollector™ and MethylCollector™ Ultra utilize a His-tagged recombinant methyl-binding protein 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 MethylCollector™ kit utilizes a recombinant His-tagged MBD2b protein that specifically binds methylated DNA. MBD2b has been found to posses one of the highest affinities for methylated DNA among the MBD proteins1. This kit can efficiently enrich for methylated CpG islands from as few as 800 cells (~5 ng DNA).
MethylCollector™ Ultra is an enhanced version of our MethylCollector™ Kit. This method is based on the Methylated CpG Island Recovery Assay (MIRA), which utilizes the high affinity of the MBD2b/MBD3L1 complex for methylated DNA2. MethylCollector™ Ultra combines the His-tagged MBD2b protein with its binding partner MBD3L1 for specific isolation of CpG-methylated DNA. This MBD2b/MBD3L1 protein complex generates a higher affinity for methylated DNA than MBD2b protein alone3. 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. Fraga, M.F. et al. (2003) Nucleic Acids Res., 31: 1765-1774
2. Rauch, T. and Pfeifer, G. (2005) Lab. Investigations, 85: 1172-1180
3. 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™ and 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. 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 either endpoint (Figure 2) or real time (Figure 3) PCR with the provided Xist (methylated promoter) and APC (unmethylated promoter) PCR primer mixes. The methylated Xist promoter gave a robust signal and showed strong enrichment in the eluted fraction. The unmethylated APC 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.
APC, adenomatosis polyposis coli, is an unmethylated promoter in healthy tissues. Methylation of this promoter is associated with several types of cancers. The region amplified by this primer pair is 338 base pairs and contains 29 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.
Figure 2: MethylCollector™ Ultra specifically binds CpG-methylated DNA.
A comparison of unbound and eluted fractions shows the specificity of the MBD2b/MBD3L1 protein complex for CpG-methylated DNA. The methylated Xist promoter was only detected in the eluted fraction, while the unmethylated APC promoter was only detected in the unbound fraction.
Figure 3: Real Time PCR analysis of MethylCollector™ Ultra
Analysis of the unbound and eluted fractions by real time PCR shows the enrichment of methylated DNA for both the Xist and NY-ESO loci, while the unmethylated APC locus is not detected in the eluted 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.
MethylCollector™ Ultra Contents & Storage
His-MBD2b/MBD3L1 protein complex, Binding Buffer AM7, Elution Buffer AM1, Protease Inhibitors, Proteinase K, Proteinase K Stop Solution, Magnetic Nickel Beads, Mse I digested Human, male genomic DNA, Xist PCR Primer Mix methylated control, APC 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™ Contents & Storage
His-MBD2b protein, Binding Buffer AM7, Elution Buffer AM1, Protease Inhibitors, Proteinase K, Proteinase K Stop Solution, Magnetic Nickel Beads, Positive and Negative Control DNA, MCJ PCR Primer Mix, PCR Buffer, Loading Dye, Bar Magnet, Glue dots and PCR tubes. Storage conditions vary from room temperature to -80°C, please see manual for specific details. All products guaranteed for 6 months from date of receipt.
