Bisulfite sequencing

In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.

The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulfite conversion (Figure 1).

All strategies assume that bisulfite-induced conversion of unmethylated cytosines to uracil is complete, and this serves as the basis of all subsequent techniques.

Alternative methods to bisulfite sequencing include Combined Bisulphite Restriction Analysis and methylated DNA immunoprecipitation (MeDIP).

A further method to differentiate converted from unconverted bisulfite-treated DNA is using high-resolution melting analysis (HRM), a quantitative PCR-based technique initially designed to distinguish SNPs.

[11] The PCR amplicons are analyzed directly by temperature ramping and resulting liberation of an intercalating fluorescent dye during melting.

The degree of methylation, as represented by the C-to-T content in the amplicon, determines the rapidity of melting and consequent release of the dye.

This method allows direct quantitation in a single-tube assay, but assesses methylation in the amplified region as a whole rather than at specific CpG sites.

The primer is allowed to extend one base pair into the C (or T) using DNA polymerase terminating dideoxynucleotides, and the ratio of C to T is determined quantitatively.

[13] However, matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry analysis to differentiate between the two polymorphic primer extension products can be used, in essence, based on the GOOD assay designed for SNP genotyping.

[14] A recently described method by Ehrich et al. further takes advantage of bisulfite-conversions by adding a base-specific cleavage step to enhance the information gained from the nucleotide changes.

This method demonstrated efficacy for high-throughput screening, allowing for interrogation of numerous CpG sites in multiple tissues in a cost-efficient manner.

This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest.

In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences.

[19] This method amplifies bisulfite-converted DNA with both methylated-specific and unmethylated-specific primers, and determines the quantitative ratio of the two products by comparing the differential peaks generated in a melting curve analysis.

[2] It is important to ensure that reaction parameters such as temperature and salt concentration are suitable to maintain the DNA in a single-stranded conformation and allow for complete conversion.

This could lead to the failure of the PCR amplification, or the loss of quantitatively accurate information on methylation levels resulting from the limited sampling of template molecules.

Thus, it is important to assess the amount of DNA degradation resulting from the reaction conditions employed, and consider how this will affect the desired amplicon.

[29][30] A potentially significant problem following bisulfite treatment is incomplete desulfonation of pyrimidine residues due to inadequate alkalization of the solution.

[2] A final concern is that bisulfite treatment greatly reduces the level of complexity in the sample, which can be problematic if multiple PCR reactions are to be performed (2006).

The advances in bisulfite sequencing have led to the possibility of applying them at a genome-wide scale, where, previously, global measure of DNA methylation was feasible only using other techniques, such as Restriction landmark genomic scanning.

It is believed that failures to produce cloned animals with normal viability and lifespan result from inappropriate patterns of epigenetic marks.

Global hypomethylation results in decreased genomic stability, while local hypermethylation of tumour suppressor gene promoters often accounts for their loss of function.

[33] This is based on a multi-tiered strategy, whereby bisulfite sequencing is used to obtain high-resolution methylation profiles for a limited number of reference epigenomes, while less thorough analysis is performed on a wider spectrum of samples.

This approach is intended to maximize the insight gained from a given amount of resources, as high-resolution genome-wide mapping remains a costly undertaking.

Figure 1: Outline of bisulfite conversion of sample sequence of genomic DNA. Nucleotides in blue are unmethylated cytosines converted to uracils by bisulfite, while red nucleotides are 5-methylcytosines resistant to conversion.
Figure 2: Outline of the chemical reaction that underlies the bisulfite-catalyzed conversion of cytosine to uracil.
Figure 3: DNA methylation analysis methods not based on methylation-specific PCR. Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination based on the changes within the amplicon as a result of bisulfite conversion.
Figure 4: Methylation-specific PCR is a sensitive method to discriminately amplify and detect a methylated region of interest using methylated-specific primers on bisulfite-converted genomic DNA. Such primers will anneal only to sequences that are methylated, and thus containing 5-methylcytosines that are resistant to conversion by bisulfite. In alternative fashion, unmethylated-specific primers can be used.