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
3. Bisulphite Deamination
Sulphonation & Hydrolytic Deamination
Desalting
Alkali Desulphonation
4. PCR Amplification
Primer Design
Primer Design Guidelines
Thermocyling
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.
Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5′-CpG-3′ dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG “islands” commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the “gold standard” for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine “C” versus thymine “T” residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.
Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5′-CpG-3′ dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG “islands” commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the “gold standard” for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine “C” versus thymine “T” residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.
Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5′-CpG-3′ dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG “islands” commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the “gold standard” for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine “C” versus thymine “T” residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.