Esophageal Adenocarcinoma Methods and Protocols

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Bisulfite conversion is the most commonly used method for study-

ing DNA methylation in which the non-methylated cytosines are

converted to uracil. The steps involve sulfonation of cytosine,

deamination and then followed by desulfonation to form uracil.

5-Methylated cytosine will not be converted to uracil and remains

unchanged. The subsequent PCR amplification involving the DNA

1.2 Bisulfite
Conversion Chemistry

Fig. 1 Schematic presentation of DNA methylation analysis. Denaturation of DNA

from patients and healthy donor was carried out followed by extraction. Then the

DNA was treated with sulfite for bisulfite modification. Bisulfite-converted DNA

was analyzed by methyl-sensitive PCR and high-resolution melt curve analysis.

Then check the PCR products in agarose gel and clone them. Finally, sequencing

of the selected PCR/HRM products was carried out to confirm the methylation

status of tested samples

Farhadul Islam et al.