253(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).- 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. - 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. - Design the primers, which can amplify the bisulfate-converted
DNA of interest using PCR with the amplicon length about
500 bp. - Check the PCR products using agarose gel electrophoresis.
- Clone the PCR products and proceed to sequencing analysis.
- Universal methylated and unmethylated DNA are used as posi-
tive controls to show the presence of methylated and demeth-
ylated DNA. - 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.- 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