Esophageal Adenocarcinoma Methods and Protocols

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(d) Incubate the mixture using a thermal cycler with the fol-
lowing condition: two cycles with 95 °C for 4 min, and
then 55 °C for 4 h; one cycle with 95 °C for 4 min, and
then 55 °C for 2 h. Keep the mixture at 4 °C.
(e) Desalt the mixture using typical DNA purification columns.
(f) Elute the DNA using 2 mM Tris (pH 8.0).


  1. The cytosine-6-sulfonate is deaminated to uracil-6-sulfonate as
    follows:
    (a) 54 μl bisulfite-treated DNA is added with 6 μl 3 M NaOH
    (see Note 3).
    (b) Incubate the mixture at 37 °C for 15 min.
    (c) Keep the mixture on ice.

  2. The uracil-6-sulfonate is desulfonated to uracil as follows:
    (a) Add 2 μl glycogen (1 μg/μl) and then 26 μl of 10 M
    ammonium acetate (pH 7.8) to the mixture in step 2 (see
    Note 4).
    (b) 500 μl ice-cold ethanol is added to the mixture.
    (c) Put the mixture in ice-water bath for 10 min.
    (d) Spin the DNA at 13,000 × g using benchtop centrifuge.
    (e) Wash the precipitate using 200 μl 70% ice-cold ethanol.
    (f) Spin the DNA at 13,000 × g for 5 min at 4 °C and then
    air-dry.
    (g) Resuspend the bisulfate-converted DNA in Tris-EDTA
    (pH 8.0) with a final concentration ~20 ng/μl.
    (h) Store the mixture at − 20 °C until use.

  3. Design the primers, which can amplify the bisulfate-converted
    DNA of interest using PCR with the amplicon length about
    500 bp.

  4. Check the PCR products using agarose gel electrophoresis.

  5. Clone the PCR products and proceed to sequencing analysis.

  6. Universal methylated and unmethylated DNA are used as posi-
    tive controls to show the presence of methylated and demeth-
    ylated DNA.

  7. Non-bisulfite-treated DNA is used as the negative control to
    show the success of bisulfite treatment on the DNA.


Preparation, DNA denaturation, and bisulfite conversion are simi-
lar as described in Subheadings 3.1.1, 3.1.2, and 3.1.3.


  1. Prepare PCR reaction mixture for MS-HRM in a 10 μl total
    volume containing 5 μl of HRM master mix, 1 μl of 40 ng/μl


3.1.4 PCR Amplification
on Bisulfite-Converted DNA
(First Approach)


3.2 MS-HRM
(Second Approach)


3.2.1 MS-HRM Analysis


DNA Methylation in Esophageal Adenocarcinoma
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