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- Gel staining reagent.
- Gel loading dye.
- Plasmid construct for cloning the PCR products.
- Universal methylated and unmethylated DNA.
- Methyl-specific primers.
- Quantitative PCR platform compatible with HRM.
- HRM kit.
- RNase free water.
- PCR tubes.
3 Methods
- DNA is extracted from fresh or frozen tissues after proteinase
K digestion using the standard phenol-chloroform method.
This step is for removing the proteins from cell lysates and gets
the DNA by precipitation. - With the known DNA sequence of the gene of interest, select
the restriction enzymes, which cut the extracted DNA (~1 μg)
into smaller fragments without cutting the sequence of inter-
est. The fragmentation of DNA can be examined by agarose
gel electrophoresis. The smaller DNA fragments are for the
subsequent bisulfite reactions as described below. - Resuspend the extracted DNA and measure the DNA amount
by spectrophotometric method. Store the DNA at − 20 °C
until use. - The digested DNA is denatured using 1/10 volume of 3 M
NaOH at 37 °C for 15 min. The DNA is strand-separated
under strong alkaline condition. - Put the treated DNA on ice instantly after the denaturation.
This is to avoid the self-annealing of the separated strands. The
double-stranded DNA reduces the yield of bisulfite conversion. - The unmethylated cytosine in the single-stranded DNA is sul-
fonated as follows:
(a) Freshly prepare 3.6 M sodium bisulfite (pH 5.0) (see Note 1)
and 10 mM hydroquinone (see Note 2).
(b) 1 ml of the 10 mM hydroquinone is added to 15 ml of the
3.6 M sodium bisulfite. Then, adjust the total volume to
20 ml with distilled water.
(c) Add 430 μl of the sodium bisulfite-hydroquinone solution
to 0.5 μg of the digested DNA and then mix by inversion
ten times.
2.5 Methyl-Sensitive
High-Resolution Melt
(MS-HRM) Curve
Analysis (for Second
Method)
3.1 PCR and
Sequencing Method
3.1.1 Preparation of DNA
3.1.2 Denaturation
of DNA
3.1.3 Conversion of DNA
Using Bisulfite
Farhadul Islam et al.