MethylEasy™ DNA Bisulphite Modification Technology

The DNA Bisulphite Modification Method

Figure 1 The DNA Bisulphite Modification MethodPrior to the early 1990s, there were very few techniques that could assess the methylation patterns in genomic DNA at the level of individual CpG sites and most of these techniques required relatively large quantities of starting DNA (up to 10μg). This difficulty was overcome by the advent of the DNA Bisulphite Modification Method conceived by Dr G W Grigg and brought to practise by Dr D Millar (now Chief Scientist of Genetic Signatures Limited) in conjunction with Dr M Frommer and colleagues [1].

The bisulphite method for determining the methylation status of cytosine residues in a DNA molecule depends on the reaction of bisulphite with cytosines in single stranded DNA. Cytosines are converted to uracils whereas 5-methylcytosines (5-mC) are unreactive (See Figure 1 and Reference 2)[2]. The modified DNA strands can be amplified by the use of the Polymerase Chain Reaction (PCR) and either sequenced directly, or cloned and sequenced to give methylation data from single DNA molecules.

Genetic Signatures Ltd has invented a new DNA bisulphite modification method, which dramatically improves the yield and the efficiency of the analysis of modified DNA. The MethylEasy™ DNA Bisulphite Modification technology has been developed as a result of this innovation.

Previous DNA Modification Methods
There are shortcomings with all previous bisulphite based methods used to determine the methylation status of any DNA molecule. Conventional bisulphite treatments utilized to date result in the loss of between 84 and 96% of the starting DNA[3], require restriction endonuclease digests, long incubation times, embedding of the DNA in agarose, multiple tube changes and/or multiple DNA purification steps.

The MethylEasy technology addresses all of the shortcomings of previous bisulphite treatments and has many advantages, including:

  • Rapid and easy to use – 90 minute total turnaround time for fully converted purified DNA
  • no DNA pre-treatment
  • virtually no loss of DNA
  • improved sensitivity
  • greater amplification efficiency
  • longer fragment generation and
  • increased stability of the template DNA (room temperature for over one month)

[1] Frommer, M., et. al., Proc.Nat.Acad.Sci.USA, 1992, 89, 1827-1831.
[2] Hayatsu, H., Wataya, Y., Kai, K., & Iida, S. Biochemistry, 1970, 9, 2858-2866
[3] Grunau, C., Clark, S. J., & Rosenthal, A. Nucleic Acids Res. 2001, 29, e65.