Basics: The bisulphite modification method has been one of the most significant developments in methylation analysis. The key advantage of this method is sensitivity, as the degree of methylation in each position of cytosines can be identified with great precision. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive (Fig. 1). The converted DNA is then amplified with specific primers (where uracil corresponds to thymine in its base pairing behaviour), followed by downstream detection techniques, such as sequencing (with or without cloning) or microarrays. All the cytosine residues remaining in the interrogated sequence represent methylated cytosines in the genome.
This protocol can also be applied for small sample sizes of a few nanograms or below. The standard protocol uses 500 ng. The eluted DNA is suited for all techniques currently used for the analysis of DNA methylation; including PCR, real-time PCR, MSP-PCR, bisulfite sequencing, COBRA, microarrays and pyrosequencing.
Fig. 1: Step 1: Sulphonation; Step 2: hydrolytic deamination and Step 3: alkali-desulphonation. Bisulfite conversion is performed under acidic conditions and preferentially deaminates cytosine in a nucleophilic attack whilst the methyl group on 5-methylcytosine is protecting the amino group from the deamination.
Yet, all bisulfite protocols have some limitations.
The most commonly encountered artifact arises due to the high salt concentrations in the bisulphite reaction, which favors reannealing of DNA and, in turn, inhibits the sulphonation. The incomplete conversion will then be detected as false methylated cytosines. In addition, a small portion of 5-methylcytosines may be converted to thymine, which results in false negatives. Furthermore, treatment with bisulphite, especially at high temperatures, leads to DNA degradation due to partial acid-catalyzed depurination. Consequently, a high proportion of the template DNA is too fragmented to be analyzed. This problem is important when only limited amounts of starting material is available, e.g. when using post-mortem brain samples, small amounts of body fluids, or microdissected tissues. This predicament intensifies further, if the remaining DNA fragments are lost during the subsequent purification (desalting), which has to be extremely stringent. Since sodium bisulphite is a very effective pH buffer, residual bisulphite will prevent the complete alkalinization of the DNA solution during desulphonation. However, if the reaction intermediate uracil-sulphonates are not converted, any DNA polymerase will be unable to replicate the template. Additionally, due to the 3-dimensional nature of the ssDNA template, it may occur that some cytosines are not converted because they are included in hairpin structures that contain double-stranded regions. When analyzing hypermethylated sequences, this effect can be even more severe, since methyl groups stabilize the double helix structure.
Fig. 2: After bisulphite treatment, all of the possible 16 permutations of a four base DNA sequence containing unmethylated C and T will become identical.
To circumvent some of the classical problems with this technology, we developed a new protocol. This protocol was developed to improved several critical steps, which leads to higher recovery rates, a higher conversion rate (99.5-100%) and a much faster procedure (under 2 hours). This method uses the presence of additional denaturing reagents and scavengers.
