The overall goal of this procedure is to enable DNA methylation analysis of CPG Dinucleotides by genomic sequencing of by sulfite converted DNA. This is accomplished by first denaturing, the double stranded DNA, so that the bisulfite conversion reaction can take place. The second step is to convert all of the unmethylated cytosines in the genomic DNA to uracil by bi sulfite deamination.
This reaction involves three phases. The first is sulfation of the cytosine residue by the addition of B sulfite to the five six double bond. Secondly, hydraulic deamination of the cytosine by sulfite derivative produces a SSL by sulfite derivative, and thirdly, removal of the sulfonate group by alkali treatment results in a SSL residue.
Next, the target region is amplified by bi sulfite conversion specific primers and PCR amplification. The final step is cloning and sequencing of the PCR product to give single nucleotide resolution for methylation across the DNA molecule. Ultimately, results show DNA methylation of CPG Dinucleotides in genomic DNA through exploiting the increased sensitivity of cytokine versus five methyl cytosine to bi sulfate deamination under acidic conditions.
The main advantage of this technique over existing methods like southern blood analysis, is that it can allow single nucleotide resolution of d methylation. This method can Help answer key questions in the epigenetics field, such as the role of DNA methylation in human diseases like cancer. To prepare DNA samples for bi sulfite conversion, first incubate two micrograms of genomic DNA with bi sulfite, DNA lysis buffer, and a total volume of 18 microliters for one hour at 37 degrees Celsius.
Then denature the genomic DNA by adding two microliters of freshly prepared three molar sodium hydroxide to a final concentration of 0.3 molar. Incubate the samples at 37 degrees Celsius for 15 minutes in a water bath, followed by incubation at 90 degrees Celsius for two minutes. In a heat block, immediately place the tubes on ice for five minutes, centrifuge the tubes for 10 seconds to 10, 000 Gs to ensure the DNA is at the bottom of the tube, the denature DNA samples are now ready for B cell fight deamination, which will be demonstrated next.
Prior to the sulfation and hydrolytic deamination reactions, prepare fresh solutions of 10 millimolar quinol and saturated sodium meta by sulfite pH 5.0. As the sodium meta by sulfite is a saturated solution, small lumps may remain undissolved. Add the sodium meta by sulfite 208 microliters and quinol 12 microliters to the denature DNA 20 microliters in a final volume of 240 microliters.
Vortex the tubes and centrifuge for 10 seconds to ensure all of the droplets are at the bottom of the tube. Overlay the samples with mineral oil. Incubate the samples at 55 degrees Celsius in a water bath for four to 16 hours, depending on the quantity and quality of the DNA to be converted.
It is important that the bisulfite conversion takes place in the dark to avoid oxidation. At the end of the appropriate incubation time, spin the tubes briefly in a micro centrifuge to ensure all of the liquid is at the bottom of the tube. Then carefully pipette out the DNA solution from the bottom of the tube without taking up any of the mineral oil.
For the next steps, our lab uses the promega wizard DNA cleanup system. Add one milliliter resin to the DNA samples, invert the tube to mix, then pipette the solution into a syringe attached to a desalting column. Finally, to remove any free bi sulfite ions, pass the bi sulfite treated DNA through the Desalting column and allude in 50 microliters of Milli Q water.
The next step is to remove the bisulfite attic from the uracil ring by desalination. To accomplish this, add 5.5 microliters of freshly prepared three molar sodium hydroxide to each bisulfite treated DNA sample for a final concentration of 0.3 molar. Then incubate the samples at 37 degrees Celsius for 15 minutes.
Centrifuge the samples briefly and then add one microliter of TRNA to each sample. Neutralize the solution by adding ammonium acetate to a final concentration of three molar. Next ethanol precipitate the DNA.
Add ice cold, 100%ethanol 330 microliters and mix well by inversion. Leave at minus 20 degrees Celsius for one hour to overnight centrifuge at 14, 000 GS for 15 minutes at four degrees Celsius. Then remove all traces of supernatant and air dry the precipitated DNA for approximately 20 minutes.
After air drying, the pellet is almost impossible to see resuspend the DNA pellet in 50 microliters of 0.1 XTE or water leave at room temperature for approximately two hours during this time, occasionally, vortex the tubes to ensure the DNA is dissolved, followed by a quick centrifuge. Once the DNA is dissolved, it can be stored at minus 20 degrees Celsius for one to 10 years, depending on the quantity and quality of DNA or be used immediately. For PCR amplification primer optimization is the most difficult aspect of this procedure.
Therefore, the following guidelines are provided to help ensure primer design success. To illustrate these guidelines, the target sequence has been by sulfite converted so that when written converted cytosines appear as blue Ts and unconverted CPG sites appear as red cgs primers should be 24 to 30 base pairs in length to ensure specificity. Primers should have a similarly predicted TM above 50 degrees Celsius and not differ by more than one to two degrees Celsius.
Primers should contain multiple approximately 25%C to T bases to ensure conversion specificity. The final base that the three prime end should be a C to T to ensure amplification of converted D-N-A-C-P-G dinucleotides should be avoided in the primer sequence to avoid potential bias towards methylated unmethylated or unconverted templates. However, if unavoidable, the cytosine in the CPG site should be replaced with a Y to help ensure unbiased amplification of converted DNA.
Finally, design nested or semi nested primer sets to ensure specificity or sensitivity of the PCR reaction if required. Prepare PCR amplification reaction mixtures in 25 microliter aliquots for optimization. Each reaction contains by sulfite converted genomic DNA DPS primers, magnesium chloride, potassium chloride tris hydrochloride pH 8.3 anac polymerase to test for amplification bias of methylated or unmethylated material.
Use a 50 50 methylated unmethylated fully bi sulfite converted control sample to test for proportional PCR amplification with bi sulfite conversion. Specific primers and PCR reaction mix in a temperature gradient thermocycler set the run reaction in a gradient plus or minus three degrees Celsius from the TM of the primer across eight to 10 tubes, vortex the tubes and spin briefly in a micro centrifuge prior to placing them in the thermocycler. This figure shows an agros gel with a temperature gradient PCR amplification profile from a mixture of 50%methylated and unmethylated DNA.
The PCR Amplicons and lanes marked with T lanes two, five and eight have been treated with a restriction enzyme that will digest only methylated DNA optimal PCR amplification conditions should amplify methylated and unmethylated amplicons in proportion and without bias resulting in an equal amount of cut and uncut PCR product as seen here. The optimal thermocycling conditions for non-biased amplification is at 55.6 degrees Celsius. Complete conversion of the bi sulfite DNA can be analyzed by digestion with a cytosine site-specific enzyme that will only digest unconverted DNA lanes three, six and nine.
Complete bi sulfite DNA conversion is indicated here by the absence of cut DNA in all three lanes, lanes three, six, and nine. This realtime dissociation plot shows an equal proportion of methylated and unmethylated DNA red orange line 50 50 mix compared to the control amplification of fully methylated pink line m and Unmethylated, DNA green line U, which dissociate at 82.9 degrees Celsius and 86.9 degrees Celsius respectively. This indicates that there is no amplification bias based on the temperature at which the different molecules will dissociate.
The resulting PCR fragments from amplification of bi sulfite treated samples can be visualized by agros gel electrophoresis and sequenced. Directly shown here is 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, where cell lines Y and Z show varying degrees of methylation as seen by overlapping GA signals shown here. Figure four A is a sequence trace from three different cell lines with CPG sites highlighted in yellow.
After direct sequencing of individual clones, the methylation state of the individual molecules can be tabulated in a bi sulfite map. To visualize the heterogeneity methylation, the density of methylation at individual CPG sites is shown in red. In this representative bi sulfite map, high throughput quantitative methylation analysis of a sample.
Using sequenome epi typer technology can produce a specific spectrum dependent on the presence of methylated cytosines, such as shown in this example Figure five A.A summary of ratios in the sample can then be extrapolated in the form of an epigram figure five B.This example shows the percentage of DNA methylation at each CPG site for four different cell lines. Finally, a methylation plot summary figure five C can be derived from the epigram After its development. This technique paved the way for researchers in the field of epigenetics to explore DNA methylation and its role in human diseases.
