Esophageal Adenocarcinoma Methods and Protocols

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  1. Gel staining reagent.

  2. Gel loading dye.

  3. Plasmid construct for cloning the PCR products.

  4. Universal methylated and unmethylated DNA.

  5. Methyl-specific primers.

  6. Quantitative PCR platform compatible with HRM.

  7. HRM kit.

  8. RNase free water.

  9. PCR tubes.


3 Methods



  1. 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.

  2. 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.

  3. Resuspend the extracted DNA and measure the DNA amount
    by spectrophotometric method. Store the DNA at − 20 °C
    until use.

  4. 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.

  5. 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.

  6. 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.
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