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DNA hypermethylated mediated loss or reduced expression of
genes involving in cell cycle regulation, DNA and growth factors
repair, metastasis, apoptosis, etc. are most prominent in esophageal
adenocarcinoma [ 4 , 5 ]. Promoter methylation of tumor suppressor
genes promotes the conversion of nonneoplastic esophageal mucosa
to Barrett esophagus—a precursor lesion that can lead to adenocar-
cinoma [ 5 – 7 ]. Subsequent losses of additional genes by mutations
and/or methylation lead the progression of dysplasia-metaplasia-
neoplasia sequence in patients with esophageal adenocarcinoma
[ 4 , 5 , 7 ]. For example, hypermethylation- mediated inactivation
of CDKN2A was noted in patients with Barrett esophagus or
esophageal adenocarcinoma [ 8 – 10 ]. In addition, hypermethylation
of CDKN2A, TIMP3, and ESR1 is associated with the onset of
Barrett esophagus or dysplasia [ 11 ]. Furthermore, promoter meth-
ylation of e-cadherin acts as a marker for the transition of dysplasia
to adenocarcinoma in patients with esophageal adenocarcinoma
[ 12 , 13 ]. Thus, methylation status of gene (s) can stratify cancer
patients with esophageal adenocarcinomas, which could be used as
a potential marker for prognosis of the disease. Table 1 shows the
significance of genes’ methylation in esophageal adenocarcinomas.
There are several methods available to check the methylation
status of DNA samples. All the methods can be divided into two
categories depending on (1) the discovery of unknown methyla-
tion changes or (2) the assessment of DNA methylation within
particular regulatory region of interest. Methylation of DNA of
particular gene of interest can be done with a number of methods,
including bead array, polymerase chain reaction (PCR) and
sequencing, pyrosequencing, methylation-specific PCR, PCR with
high- resolution melt curve analysis, etc. In this chapter, we describe
PCR and sequencing based protocol followed by bisulfite conver-
sion of the DNA obtained from patients with esophageal adeno-
carcinoma and healthy controls. The protocol includes five steps:
(1) preparation of DNA, (2) denaturation of DNA, (3) bisulfite
conversion of DNA, (4) PCR amplification of bisulfite- converted
DNA, and (5) confirm the methylation status of DNA by sequenc-
ing. In addition, screening of DNA methylation with methyl-sen-
sitive high-resolution melt (MS-HRM) curve analysis is illustrated.
Figure 1 illustrates the flow diagram of the experimental protocol
for methylation of DNA samples.The methylation study on DNA can be used to understand the
epigenetic mechanisms on gene expression. The methyl group (an
epigenetic factor) can tag DNA and control (activation or suppres-
sion) gene expression. The binding of epigenetic factors can change
how DNA is wrapped around histone protein and the availability
of gene expression of the involved DNA.
The epigenetic modification of DNA in mammalian cells involves
the methylation of cytosine nucleotides. The primary target sequence1.1 Principle
and Background
of Methylation
Followed by Bisulfite
Conversion
Farhadul Islam et al.