The Methylamp DNA Modification Kit is a complete set of optimized reagents designed to convert DNA so that 5-methylcytosines can be detected in downstream applications for gene-specific DNA methylation analysis. The kit has the following advantages and features:
- Total protocol time is less than 2 hours.
- 99.9% conversion rate of unmethylated cytosine to uracil/thymine
- Extremely low degradation of DNA in the modification process, avoiding more than 90% of DNA loss.
- It requires only 50 pg of input DNA, equivalent to about 20 cells.
- Extremely simple, robust, and reliable sodium bisulfite conversion protocol.
- Suitable for bisulfite sequencing (direct and cloning), next-generation sequencing, pyrosequencing, PCR (real-time, end-point, methylation-specific), COBRA (combined bisulfite restriction analysis), and methylation-based microarrays.
- Highly compatible with Illumina-based workflows.
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine. DNA methylation is essential for the development of many species and is often associated with many key biological processes, as well as gene regulation. Various methods are used to assess DNA methylation states.
However, only bisulfite conversion of genomic DNA, followed by PCR amplification, cloning, and sequencing of individual PCR amplimers, provides reliable information on the methylation states of individual cytosines in individual DNA molecules. By treating DNA with sodium bisulfite, cytosine residues are deaminated to uracil leaving 5-methylcytosine intact, providing researchers with distinguishable methylated positions in the DNA sequence during subsequent application.
Principle and procedure
The DNA is chemically denatured to allow the sodium bisulfite reagent to react specifically with the single-stranded DNA, thereby determining the cytosine and creating a uracil residue. The unique DNA protection reagents contained in the Modification Buffer can prevent chemical and thermophilic degradation of DNA during the sodium bisulfite treatment process.
The included non-toxic modified DNA capture buffer allows DNA to bind tightly to the column filter, allowing DNA cleanup to take place on the column to effectively remove sodium bisulfite and salts residuals. The modified DNA can then be eluted for use in various methylation analysis applications or stored at -20 ° C for up to 2 months.