DNA Methylation: Bisulphite Modification and Analysis

Bisulphite conversion protocol

A standard protocol for the conversion of 2 μg of genomic DNA is described below. Smaller or larger amounts of genomic DNA (100 pg-200 μg) can also be bisulphite treated successfully. These reaction conditions result in complete conversion (99.5-99.7%) of almost every target DNA sequence³.

1. DNA Preparation

Prepare samples by incubating genomic DNA with bisulphite DNA Lysis Buffer (2 μg tRNA, 280 ng/μl Proteinase K, 1% SDS) in a total volume of 18 μl for 1 hr at 37°C. Note: This is important to achieve maximal bisulphite conversion, especially with DNA isolated from clinical samples where there may still be protein associated with the DNA.

2. DNA Denaturation

1. Denature 2 μg DNA in a volume of 20 μl by adding 2 μl of freshly prepared 3M NaOH to a final concentration of 0.3M.
2. Incubate the samples at 37°C for 15 min in a water bath, followed by incubation at 90°C for 2 min in a heat block. Immediately place the tubes on ice for 5 min.
3. Centrifuge the tubes at 4°C for 10 s at 10,000 g, to ensure the DNA is at the bottom of the tube.

3. Bisulphite Deamination

Sulphonation & Hydrolytic Deamination

1. Prepare fresh solutions of 10 mM Quinol and saturated sodium metabisulphite pH 5.0 (7.6 g Na₂S₂O₅ with 464 μl of 10 M NaOH, made up to 15 ml with water). The solution of saturated sodium bisulphite is achieved by gently inverting the reagent/H₂O mixture, with minimum mixing and aeration. If necessary, adjust the pH with 10 M NaOH, for full dissolution of the metabisulphite. Note: As it is a saturated solution, small lumps may still remain undissolved.
2. Add 208 μl of the saturated metabisulphite and 12 μl of 10mM Quinol to the denatured DNA (20 μl), in a final volume of 240 μl to a final concentration of 2.31M bisulphite/0.5mM Quinol, pH 5.0. Gently mix all reagents and centrifuge for 10 sec to ensure all of the droplets are at the bottom of the tube.
3. Overlay the samples with 200 μl of mineral oil. Incubate the samples at 55°C in a water bath, for 4-16 hrs. The length of bisulphite treatment is dependant on the quantity and quality of the DNA to be converted. For DNA of poor quality or degraded DNA, limit the incubation time to 4 hrs. Note: it is important that that the bisulphite conversion takes place in the dark to avoid oxidation, so if that is not possible wrap the tubes in foil prior to incubation.
4. At the end of the appropriate incubation time, spin the tubes briefly in a microcentrifuge to ensure all the liquid is at the bottom of the tube.
5. Recover the bisulphite treated DNA from under the oil layer by carefully pipetting out the DNA solution from the bottom of the tube, without taking up any of the mineral oil.

Desalting

5. Remove any free bisulphite ions by passing through a desalting column and eluting in 50 μl of milli-Q water (MQH₂O). Depending on the quantity and quality of the genomic DNA and how it is prepared, different desalting columns can be used. We routinely use Promega Wizard Clean up columns, as these columns are suitable to remove the SDS used in the preparation of the DNA prior to conversion.

Alkali Desulphonation

6. The bisulphite adduct is removed from the uracil ring by desulphonation. Desulphonate by adding 5.5 μl of freshly prepared 3M NaOH to a final concentration of 0.3M, then incubate the samples at 37°C for 15 min.
7. Centrifuge briefly and add 1 μl of tRNA (10 mg/ml).
8. Neutralize the solution by the addition of 33 μl of ammonium acetate (NH₄O Ac), pH 7.0 to a final concentration of 3M.
9. Ethanol-precipitate the DNA by adding 330 μl of ice cold 100% ethanol, mix well by inversion. Leave at -20μC for 1 hr to overnight. Centrifuge at 14,000 g for 15 min at 4°C. Remove all traces of supernatant and air dry the precipitated DNA for ~20 min.
10. Re-suspend the DNA pellet in 50 μl/μg of 0.1 TE (10mM Tris-HCL, 0.1mM EDTA, pH 8.0) or H₂O. Leave at room temperature for approximately 2hrs. Occasionally vortex the tubes to ensure the DNA is dissolved, followed by a quick centrifuge.
11. Use immediately for PCR amplification, or store at -20°C for 1-10 years depending on the quantity and quality of DNA.

4. PCR Amplification

Primer Design

1. Effective design of PCR bisulphite conversion-specific primers is crucial for ensuring that efficient, unbiased amplification of completely converted DNA occurs. The following guidelines are used to aid primer design.

Primer Design Guidelines

Thermocyling

2. To test for proportional PCR amplification of methylated and unmethylated DNA, use a 50:50 Methylated/Unmethylated fully bisulphite converted control sample and amplify with the bisulphite conversion-specific primers under the optimized PCR reaction conditions (Figure 3). For the 50:50 Methylated:Unmethylated control, use in vitro SssI methylated DNA and either whole genome amplified (WGA) DNA or whole blood DNA. Please be aware that some genes will be naturally methylated in blood and therefore in these cases it is best to only use WGA DNA as the “Unmethylated” control.
3. Prepare PCR amplification reaction mixtures in 100 μl aliquots containing 2 μl of bisulphite converted genomic DNA, 200 μM dNTP’s, 1 μM primers, 1.5 mM MgCl₂, 50 mM KCl, 10mM Tris-HCL, pH 8.3 and 0.15 μl Taq polymerase.
4. In a temperature gradient thermocycler, set the run reaction in a gradient +/- 3°C from the predicted Tm of the primer across 10 tubes

5. To test that the methylated and unmethylated amplicons have been amplified in proportion, the amplicon can be digested with an informative restriction enzyme, such as Taq 1 (TCGA), that will digest methylated DNA but will not digest unmethylated DNA when the restriction enzyme site is altered after bisulphite conversion to TTGA. The extent of digestion can be visualized by agarose gel electrophoresis (Figure 3A). Alternatively, SYBRGreen (0.2 μl of 1:1000 dilution) can be added to the PCR reaction and the extent of methylation can be assessed by performing heat dissociation plots in a real-time PCR thermocycler (Figure 3B).
6. PCR Reaction mix including MgCl₂ concentration and thermocycling conditions may need to be adjusted to increase PCR efficiency or to ensure proportional PCR amplification of methylated and unmethylated PCR fragments.
7. Once the PCR conditions are optimized, PCR amplification can be performed on bisulphite converted samples. Note: Once the Tm is optimized a temperature gradient PCR need not be used.

5. Representative Results:

An example of bisulphite PCR amplification optimization is shown in Figure 3. Optimal PCR amplification conditions should amplify methylated and unmethylated amplicons in proportion and without bias. Figure 3A shows an agarose gel with a temperature gradient PCR amplification profile from a mixture of 50% methylated and unmethylated DNA. The PCR amplicons have been treated with a restriction enzyme, Taq 1 (TCGA), that will digest methylated (M) DNA but will not digest unmethylated (U) DNA as the restriction enzyme site is altered by bisulphite conversion to TTGA. An equal amount of cut and uncut PCR product is expected if methylated and unmethylated DNA is amplified in proportion. It can be seen on this gel that the optimal thermocyling conditions for non-bias amplification is at 56.1°C (T). Complete conversion of the bisulphite DNA can also be analyzed by digestion with cytosine-site specific enzyme such as HpaIII (CCGG). HpaIII will only digest if the conversion has failed, as the restriction site will be maintained. If the conversion is complete the restriction site will be converted to TCGG or TTGG depending on the methylation state of the DNA. Complete bisulphite DNA conversion can be seen in Figure 3A (H).

Figure 3B shows a real time dissociation plot that can also be used as a tool to determine whether there is any amplification bias based on the temperature at which the different molecules will dissociate. In this figure it can be seen that the PCR has amplified methylated (M) and unmethylated (U) DNA in proportion from a mix of 50% methylated and unmethylated DNA compared to the control amplification of fully methylated and unmethylated DNA which dissociate at 82.1μC and 78.9μC respectively.

After optimization of thermocyling and reaction conditions Bisulphite treated samples can be amplified with strand specific and bisulphite specific primers in either a single or semi nested PCR reaction. The resulting PCR fragments can be visualized by agarose gel electrophoresis and sequenced either directly (Figure 4A) or by cloning and sequencing. After cloning and sequencing the methylation state of the individual molecules can be tabulated, in a bisulphite map (Figure 4B), to visualize the heterogeneity of methylation.

High throughput quantitative methylation analysis can be preformed using technology such as that utilized by the Sequenom EpiTYPER method. Using this method, bisulphite converted DNA is amplified with bisulphite specific PCR primers and followed by a proprietry cleavage process. The resulting fragments are quantitated by MALDI-TOF mass spectrometry with the specific spectrum dependent on the presence of methylated cytosines (Figure 5A). A summary of methylation ratios in the sample can then be extrapolated in the form of an Epigram (Figure 5B) or methylation plot (Figure 5C).

Figure 1. Methylated CpG schematic. In the normal cell, promoter-associated CpG islands are predominantly unmethylated (grey) whereas CpG sites within gene bodies are sparse and generally methylated (red). The panel on the right expands the molecular structure of DNA at an individual CpG site and shows methylation with a CH3 molecule at carbon 5 of cytosine.

Figure 2. Chemical conversion schematic. Analysis of DNA methylation includes four main stages as shown; denaturation, bisulphite conversion, PCR amplification and analysis. In the right panel, modifications to the cytosine molecule that occur during bisulphite conversion are depicted.

Figure 3. Optimisation of PCR amplification. A. Agarose gel electrophoresis with a temperature gradient PCR amplification profile from a mixture of 50% methylated and unmethylated DNA. Fragments have been digested with either Taq1 (T) or HpaIII (H) which digest methylated DNA and non bisulphite converted DNA respectively. B. Heat dissociation curve analysis shows an equal proportion of methylated and unmethylated DNA (red line 50/50 mix) compared to the control amplification of fully methylated (pink line, M) and unmethylated DNA (green line, U) which dissociate at 82.1°C and 78.9°C respectively.

Figure 4. Example of direct sequencing and a bisulphite map. A. Sequence trace from three different cell lines with CpG sites highlighted in yellow. Cell line X displays 100% methylation at all three CpG sites whereas cell lines Y and Z show varying degrees of methylation as seen by overlapping G/A signals. B. Representative bisulphite map showing the density of methylation (red dots) at individual CpG sites, as determined by direct sequencing of individual clones.

Figure 5. High throughput DNA methylation analysis using Sequenom. A. Spectrum view using Sequenom epiTYPER technology. DNA fragments display specific spectra, depending on amount of DNA methylation present. B. Epigram summary of the percentage of DNA methylation at each CpG site for four different cell lines. C. Methylation plot summary derived from the Sequenom Epigram.