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Detection of DNA methylation

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by

Eric Attack

on 20 July 2014

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Transcript of Detection of DNA methylation

Methods - Focus
Further methods
Methylated DNA immunoprecipitation sequencing -> antibodies

In vitro treatment with H3\C14 S-adenosyl methionine and Methyl transferase to identify global methylation levels

HPLC,UV-V detection or LC-ESIMS for determining methylated cytosine or anyother methylated bases
Coprecipitation using MeC binding proteins
Introduction
Genome of many animals, plants and fungi: methylation of DNA cytosine (C5)


Significant regulatory factor in plants


Most important epigenetic alteration of gene expression: gene silencing


Plants: highest level of DNA methylation of all eukaryotes (up to 50%)


Context:
Animals: CpG
Plants: CpNpG
Bisulfite sequencing
Has been frequently used, most common method

DNA is treated with (sodium) bisulfite




Unmethylated C to U
Methylated C unaffected


Microarray slide is coated with ssDNA probes
Denatured fragmented DNA from the plant are hybridised to the probes
Overview
Detection of DNA methylation
in plants -
Insight into methods

Restriction enzyme methods
1) Introduction

2) Methods - Focus

3) Further methods
1. DNA denaturation
-> C on ssDNA can be attacked

2. Bisulfite treatment

3. Amplification


Microarray method
Anti 5Me cytosine antibodies will bind specifically to the 5Me cytosine in the ssDNA overhangs
Fluoroprobe labelled anti Fc antibodies will enable detection of the anti 5Me cytosine antibodies
Advantages-
Since the DNA probe loci in the microarray is known, the gene that is being methlyated will become known.
Once establised quick to run
Methylation levels can be determined by BATMAN on a gene levels bases
Disadvanages-
Methylated bases can not be determined
Time consuming to develope the probes


Microarray analysis with the artifically synthesised DNA.
Similarly for artifically synthesised DNA with methyltransferase treatment to methylate Cytosine
To test for Cross reactivity-

Anti 5MeC antibodies are specific
met-specific PCR
Methylated-specific primers

Amplification only in presence of mC

Melting curve analysis -> amplification of both strands
non-met-specific PCR
Sequencing using sequencing primers mC -> C and C -> T

High resolution melting: fluorescent-labeled primers -> change in fluorescence

MALDI-TOF

Problems
5-hydroxymethylcytosine

Incomplete denaturation
HELP Assay
(
H
paII tiny Fragment
E
nrichment by
L
igation mediated
P
CR )
Two restriction enzymes -HpaII and MspI = Isoschizomers
MspI - methylation insensitive
HpaII - methylation sensitive
To compare amounts and specificity - microarray can be used
Restriction Digestion
+
Southern Blotting
Gene specific detection
Similar to HELP assay
Full transcript