Bisulfite Conversion of Genomic DNA: A Method to Study DNA Methylation in Genomic DNA Samples from Gastrointestinal Cancer Tissue
成績單
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.
