Title;

Detection of Methylated sites on

DNA .

Shehzad Ahmad
2016-UAM_39
• DNA methylation
• The method is based on natural degradation
process ,methylated cytosine are degraded into
thymine ,where un methylated cytosine are
degraded into uracil.

• Methylation sensitive single nucleotide primer

Extension Assay which uses internal primers
annealing straight of nucleotide to be detected.
 Methods of methylation
• HRMA
• It is a post PCR analytical technique. The target
DNA is treated with sodium bisulfite ,which
chemically converts unmethylated cytosine into
uracil , while methylated cytosine are preserved.
• Methyle Sensitive Southering Blotting is
simmilar to help assay, although using southern
blotting technique to probe gene specefic
differences in methylation using restriction digets
• Chip-on-Chip assays, which is based on ability
of commercially prepared anitibodies to bind
to Dna methylation-associated protiens like
MeCP2.
• Restriction landmark genomic scanning,is a
technique based upon restriction enzyme,
differential recognition of methylated and
unmethylated CpG sites;the assay is simmilar
in concept to HELP assay.
• The HELP assay, which is based on restriction
enzymes, differential ability to recognize and
cleave methylated and unmethylated CpG
DNA sites.
• GLAD-PCR ASSAY, which is based on new type
of enzymes-site-specific methyle-directed
endonucleases, which hydrolyze only
methylated DNA.
 Detection
• DNA methylation can be detected by following
assays currently used in scientific research.
• Mass spectrometry is very sensitive and
reliable method to detect DNA
methylation.MS in general is however not
informative about sequence context of
methylation , thus limites in studying function
of this DNA modification.
• Methylation-Specific PCR(MSP),which is
based on chemical reaction of sodium bisulfite
with DNA that converts unmethylated
cytosine of CpG dinucleotide to uracil or
UpG, followed by traditional PCR.However
methylated cytocine will not be converted in
this process.