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ASSIGNMENT 1
SUBMITTED TO MAAM HUMA
JAVERIA MUSHTAQ
BIOINFORMATICS
[Document subtitle]
Elaborate methods designed for detecting DNA
methylation. Discuss drawbacks of each method.
What is DNA Methylation
DNA of most organisms is modified by a post replicative process and
this results in three types of methylated bases in DNA C5
methylcytosine, N4 -methylcytosine, N6 methyladenine. This
modification is called DNA methylation.

There are several computational and biological

methods to detect DNA methylation.
Restriction endonuclease digestion, isotope incorporation and TLC
Methylation sensitive restriction enzymes.
HPLC, Mass spectrometry
Bisulphite sequencing
RP-HPLC
Radiolabelling
Antimethylcytosine
Sanger Sequencing

PCR and sequencing method

This method is used to specify DNA methylation of specific regions of
interest. Bisulphite converted DNA can be used for amplification of
region of interest followed by sequencing. Primers are used for PCR
amplification of Bisulphite converted DNA.

ADVANTAGES
Through this method we can detect the methylation status of CpG
sites in the CpG island of interest. When differences in methylation
between samples are large greater than 50 percent direct
sequencing of PCR product is obtained.

Disadvantages
To overcome the problem of unspecific amplification nested PCR is
required. Complexity of DNA is reduced due to which primer design
and amplification are problametic due to which amplification of long
fragments become difficult.

Bisulphite sequencing
This is use of bisulfite treatment of DNA before routine sequencing
to determine pattern of DNA methylation.

Advantages
Treatment of DNA with bisulfite convert cytosine into uracil and
leaves cytosine methyl residues unaffected. This method involves
specific changes in DNA sequence and it depends on cytosine
residues. By this analysis we can differentiate between single
nucleotide polymorphisms cytosine and thymidine.

DISADVANTAGES
Bisulfite sequencing cannot differentiate between 5 methylcytosine
and 5 hydroxymethyl cytosine.Bisulfite sequencing relies on the
conversion of every single unmethylated cytosine residue to
uracil this leads to incomplete inconversion. One of its
disadvantage is degradation of DNA during bisulfite treatment .
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