positive identification of unmethylated CpGs in a quick magnetic assay
HypoMethylCollector™ is a novel technology that allows for positive identification of unmethylated DNA fragments*. The kit specifically enriches for unmethylated CpG islands using a CXXC binding domain from mouse Mbd1. HypoMethylCollector™ allows researchers to identify hypomethylated promoters from normal or diseased samples. This simple and sensitive technique provides DNA suitable for use in many downstream applications, such as real time or endpoint PCR analysis of the methylation status of particular loci, sequencing, or amplification and labeling for microarray analysis.
- Fast magnetic bead based protocol is completed in less than 3 hours
- Optimized reagents and protocol ensure successful capture from DNA fragments containing only a single unmethylated CpG dinucleotide.
- Specifically detects unmethylated CpGs from 10 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
The HypoMethylCollector™ Kit uses a His-tagged CXXC domain from mouse Mbd1 to specifically bind unmethylated CpG islands of genomic DNA fragments that have been prepared by sonication or enzymatic digestion. The kit provides two binding buffers; a low-salt buffer for use with samples containing less than 5 unmethylated CpG dinucleotides and a higher salt buffer for efficient binding of samples with a large number of unmethylated CpGs. The His-CXXC protein is added to the DNA fragments, and these protein-DNA complexes are captured with nickel-coated magnetic beads. Subsequent wash steps are performed using an optimized buffer and the included magnet to separate the unmethylated fragments from the rest of the genomic DNA. The unmethylated DNA is then eluted from the beads in the presence of high salt. Following clean up, the eluted DNA is ready for use in PCR analysis or other downstream applications. See Flow Chart of the HypoMethylCollector™ process below.
Due to the high efficiency of HypoMethylCollector™ and the enormous amplification capability and specificity of PCR, analysis of the methylation status of a specific genomic DNA locus can be performed on DNA isolated from less than 1600 cells (~10 ng DNA). The specificity of the CXXC domain is able to enrich for DNA fragments containing only a single unmethylated CpG.
Flow Chart of the HypoMethylCollector™ process.
Active Motif's HypoMethylCollector™ Kit offers a novel technology for the positive identification of hypomethylated CpG DNA. The ability of the recombinant His-CXXC protein to specifically bind and enrich for unmethylated DNA is confirmed through the use of real-time and endpoint PCR analysis. The data shown below confirms the sensitivity and specificity of the assay.
Figure 1: Real-time PCR analysis of the control male DNA with unmethylated and methylated promoters.
Figure 2: Real-time PCR analysis of HypoMethylCollector™ using high salt (high stringency) binding conditions.
Figure 3: Real-time PCR analysis of HypoMethylCollector™ using low salt (low stringency) binding conditions.
Figure 4: Bisulfite sequencing confirms the specific isolation of unmethylated CpG DNA.
Figure 5: HypoMethylCollector™ can be used to compare the methylation status of various loci.
Figure 6: Endpoint PCR analysis of methylated and unmethylated promoters.
Figure 7: HypoMethylCollector™ can specifically detect a single unmethylated CpG per DNA fragment.
Figure 8: Sensitive detection of unmethylated DNA from as little as 10 ng.
HypoMethylCollector™ Contents & Storage
His-CXXC protein, Binding Buffer AM8, Binding Buffer AM9, Elution Buffer AM3, Protease Inhibitors, 1 M DTT, Magnetic Nickel Beads, Mse I digested human, male genomic DNA, GAPDH PCR Primer Mix unmethylated control, Xist PCR Primer Mix methylated 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.