This article discusses the process of DNA methylation and its role in gene expression regulation. It outlines a method for studying DNA methylation in cancerous cells using bisulfite treatment and PCR amplification.
DNA methylation involves methyl group addition to cytosine residues of DNA to form methyl-cytosine, causing DNA alteration and gene expression regulation.
To study DNA methylation, begin by taking crude DNA extract from cancerous cells. Add a dilution buffer and incubate at room temperature. The buffer dilutes the mixture and denatures the double-stranded DNA into single-stranded DNA, favoring the accessibility of bases for subsequent reactions.
Add a conversion reagent and incubate in the dark. Sodium bisulfite in the reagent triggers the sulfonation and subsequent deamination of unmethylated cytosine residues, resulting in uracil-sulfonate formation. However, methylated cytosine residues remain unchanged.
Next, supplement the DNA with a binding buffer and transfer it to a pre-assembled spin filter column. The buffer helps the DNA bind to the filter.
Centrifuge to remove the buffer and bisulfite reagent mix from the trapped DNA in the column. Discard the flow-through. Add an alkaline desulphonation buffer to the DNA. The buffer aids in sulfite group removal from uracil-sulfonate to form uracil.
Centrifuge to remove the spent buffer. Now, add an elution buffer to the column to elute the converted DNA. Perform PCR to amplify the DNA. All converted uracil residues amplify as thymines, while the methylated cytosine residues amplify as cytosines.
Sequence cytosine residues of the amplified DNA to generate a methylation profile of the sample.
Perform the bisulfite treatment on 45 microliters of the digested tissue using a bisulfite conversion kit according to the manufacturer's instructions. Add 5 microliters of dilution buffer to the sample and incubate it at 37 degrees Celsius for 15 minutes. Meanwhile, prepare the bisulfite conversion reagent by adding 750 microliters of distilled water and 210 microliters of dilution buffer to one tube of CT conversion reagent. Mix the tubes by vortexing for 10 minutes. Then, add 100 microliters of the prepared CT conversion reagent to each sample and mix by inversion.
Incubate the samples in the dark at 50 degrees Celsius for 12 to 16 hours. After the incubation, place the samples on ice for 10 minutes. Add 400 microliters of binding buffer and mix each sample by pipetting up and down. Load each sample into a spin column and place the column into a 2-milliliter collection tube. Centrifuge the samples at full speed for 1 minute and discard the flow through. Add 200 microliters of wash buffer to each column and spin at full speed for 1 minute, then discard the flow-through.
Add 200 microliters of desulfonation buffer to each column and allow the columns to stand at room temperature for 15 minutes. After the incubation, spin the columns at full speed for 1 minute and discard the flow through. Wash the column twice with 200 microliters of wash buffer, centrifuging for 1 minute at full speed after each wash.
Add 46 microliters of distilled water to each column and place it in a new sterile 1.5-milliliter single-use polypropylene tube. Spin the tubes for 2 minutes to elute the DNA and discard the column. The DNA is now ready for the analysis. Use the bisulfite modified DNA as a template for quantitative methylation-specific PCR to evaluate methylation of the promoter region in each gene analysis. Combine the reagents as described in the text manuscript, and use a 96-well real-time PCR instrument to run the thermal cycling protocol.
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