- 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.
200 ng of human, male genomic DNA was Mse I digested and tested in the HypoMethylCollector™ Kit. Eluted DNA was analyzed using PCR primers for both methylated and unmethylated promoters. The GAPDH, glyceraldehyde-3-phosphate dehydrogenase, promoter is unmethylated in healthy tissues. The early amplification of DNA in the eluted fraction verifies GAPDH as an unmethylated promoter. The Xist promoter is a methylated promoter in males and will not bind to the His-CXXC protein. This is shown with the early amplification of DNA in the unbound fraction.
Figure 2: Real-time PCR analysis of HypoMethylCollector™ using high salt (high stringency) binding conditions.
A comparison of unbound and eluted fractions under high salt binding conditions shows the specificity of the CXXC protein complex for unmethylated DNA. The unmethylated GAPDH promoter shows a 10-fold enrichment of methylated DNA in the eluted fraction as compared to the unbound fraction, while the methylated Xist promoter was mostly detected in the unbound fraction.
Figure 3: Real-time PCR analysis of HypoMethylCollector™ using low salt (low stringency) binding conditions.
A comparison of unbound and eluted fractions under low salt binding conditions shows the specificity of the CXXC protein complex for unmethylated DNA. The unmethylated GAPDH promoter shows a 10-fold enrichment of methylated DNA in the eluted fraction as compared to the unbound fraction, while the methylated Xist promoter was mostly detected in the unbound fraction.
Figure 4: Bisulfite sequencing confirms the specific isolation of unmethylated CpG DNA.
The HypoMethylCollector™ Kit was used to enrich for hypomethylated CpG islands. Following clean up, eluted DNA was used in Active Motif's MethylDetector™ Kit (Cat. No. 55001) to perform a bisulfite conversion. Converted DNA was amplified by PCR. The gel extracted PCR product was cloned and 8-10 colonies were selected for sequencing. Of the eight colonies sequenced for the APC locus, only one clone contained a single methylated CpG of the 19 CpG sites within the primer region. Of the ten colonies sequenced for the p48 locus, five clones contained methylated CpGs; none of the clones contained more than three methylated CpGs out of the 22 CpG sites within the primer region. This confirms the strong specificity of the CXXC domain for capturing and enriching for unmethylated DNA fragments.
Figure 5: HypoMethylCollector™ can be used to compare the methylation status of various loci.
Real time PCR results show the enrichment of hypomethylated CpG islands using the HypoMethylCollector™ Kit. The unbound and eluted fractions were analyzed to determine the methylation status of various loci. The methylated Xist locus is detected in the unbound fraction. The unmethylated APC and p48 loci show a specific enrichment of unmethylated DNA in the eluted fraction, with almost no detectable signal from the unbound fraction.
Figure 6: Endpoint PCR analysis of methylated and unmethylated promoters.
HypoMethylCollector™ was performed on 200 ng of the Mse I digested human, male genomic DNA that is included in the kit. Both unbound and eluted fractions were collected and analyzed in PCR. The p48, APC and Xist loci were amplified for 36 cycles, while the NY-ESO locus was amplified for 30 cycles. Results show the specific capture and elution of unmethylated DNA from the His-CXXC protein, while methylated DNA is mostly in the unbound fraction.
Figure 7: HypoMethylCollector™ can specifically detect a single unmethylated CpG per DNA fragment.
PCR products of the 14-3-3 promoter were generated with a specific number of CpG sites per fragment. Half of the PCR product was artificially methylated with Sss I. The 0.1 mg/ml DNA stocks were diluted 1600-fold and 2 µl were used in the HypoMethylCollector™ Kit in the presence of 0.05 µg/µl polydAdT with both Binding Buffer AM8 and AM9. The unbound and eluted fractions were cleaned and analyzed using endpoint PCR. The HypoMethylCollector™ Kit can enrich for unmethylated DNA fragments that contain only a single unmethylated CpG using Binding Buffer AM9. For fragments that contain five or more unmethylated CpGs, Binding Buffer AM8 is recommended.
Figure 8: Sensitive detection of unmethylated DNA from as little as 10 ng.
The sensitivity of the HypoMethylCollector™ Kit was determined by running the assay using either 100, 50, 25, 10, 5, 2.5 or 1 ng of Mse I digested DNA. The kit is able to enrich for unmethylated DNA with only 10 ng of starting material.