Esophageal Adenocarcinoma Methods and Protocols

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of interest can identify the methylation status compared with the

controls. The result of PCR amplification of the bisulfite-converted

DNA is that the methylated cytosines in the original DNA sample

are shown as C (cytosine) and the unmethylated cytosines are read

as T. The incomplete bisulfite conversion, leading to poor results,

can be due to several reasons. First, not all the unmethylated cyto-

sine residues are deaminated in the target sequence. Second, the

target DNA may be degraded under the experimental conditions.

Third, the target DNA sequence may have high percentage of

CG-rich regions, leading to the poor results of DNA sequencing.

2 Materials

Use analytical grade chemicals and reagents and prepare solutions/

buffer using ultrapure water. Prepare and store all the reagents and

buffer solution at room temperature unless otherwise indicated.

1. DNA extraction kit.


2. Proteinase K.


3. Chloroform.


4. Restriction enzymes.


5. RNase DNase free water.


6. 1.5 ml microcentrifuge tubes.


7. 3 M NaOH.


8. 1.5 ml microcentrifuge tubes.


9. RNase DNase free water.


10. Incubator.


11. 10 mM hydroquinone.


12. Molecular biology grade water.


13. 3.6 M sodium bisulfite.


14. Thermal cycler.


15. DNA purification column.


16. 2 mM Tris (pH 8.0).


17. Glycogen.


18. Ice-cold ethanol.


19. Benchtop centrifuge machine.


20. Ethylenediaminetetraacetic acid (EDTA).


21. Primer pair for gene(s) of interest.


22. Agarose powder.


23. 100 base-pair DNA ladder.

2.1 Preparation
of DNA

2.2 Denaturation
of DNA

2.3 Bisulfite
Conversion of DNA

2.4 PCR
Amplification
and Sequencing

DNA Methylation in Esophageal Adenocarcinoma