1 Internship DNA methylation of - PowerPoint Presentation

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Internship DNA methylation of λ-DNA Objective: To represent de novo methylation of DNA Model for epigenetic changes in DNA

Methylase

DNA

Cytosin

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Methyltransferases transfer a methyl group to the DNA

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Internship DNA-Methylation of

λ

-DNA Materials:

CpG

-Methyltransferase

ClaI λ-DNA

Model-DNA

DNA-Methyl-

transferase

Restriction

enzyme

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epigenetic

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Lambda is a bacteriophage of Escherichia coli.

The double-stranded DNA of the virus is linear and has a length of 48502 bp.

The genomic DNA of the phage λ is propagated in E. coli strain GM2163 and isolated from it. The λ-DNA is unmethylated because the strain GM2163 has a mutation in thegene for CpG methylase which means that the bacteria can`t conduct Cytosine methylation reactions.

DNA methylation

of λ-DNA

Material: λ-DNA (non-methylated

λ

-DNA)

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1. „Methylases, can play the role of a “molecular vaccine” by defending the genome against parasitism

by a restriction-modification gene complex” (Takahashi et la. 2002).

DNA methylation by methyltransferase

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https://pubs.niaaa.nih.gov/publications/arcr351/6-16.htmhttps://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation

The CpG methylases of eukaryotes (e.g. human CpG methylase) transfers a methyl

group to position 5 of the cytosine within the DNA sequence CG (also called CpG).The restriction enzyme

ClaI is methylation-sensitive and does not cut the recognition sequence ATCGAT if it is methylated at the C.

DNA methylation by CpG methyltransferase

e

e

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DNA methylation by

CpG

methyltransferase

Epigenetic role in the methylation of CpG sequences ("CpG islands") in promoter regions.

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Experiment Instruction1. Methylation reaction with CpG methylase

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L =

λ

-DNA, unmethylated CMT = CpG-methylase SAM = S-Adenosyl-methionine

B2 = 10 X NEB-buffer 2H2O = destilled water

You will find filled tubes with the following designations prepared in ice:

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1. DNA methylation with CpG methylase

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1.1 Label an

empty tube with “LC“ (LC = λ-

DNA CpG methylated)1.2 Fill the tube LC according to following pipetting scheme:

Component

Reaction volume

Incubation

Inactivation

Application

5 min on ice and centrifuge briefly

μl

Λ

-DNA

unmethylated

S-

adenosyl

-

methionine

-dilution (SAM),

1,6

mM

λ

-

DNA

unmethylated

(L), 0,3

μg

/

μl

H

2

O

distilled

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2. Restriction with ClaI of CpG-methylated λ-DNA

Intership epigenetic

L =

λ

-DNA unmethylated LC = λ-DNA, CpG methylatedC = Cla

I CB = 10 X cut smart buffer H2O = distilled

water

You will find filled tubes with the following designations on ice:

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2. Restriction with ClaI of CpG-methylated l-DNA

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2.1 Label 3

empty tubes with K, 1 and 22.2 Pipette according the following pipetting

scheme: Compound

Reaction volume

IncubationTubes team

CMT

λ-DNA

unmethylated

(L), 0,3

μg

/

μl

λ

-

DNA

methylated

(LC), 0,075

μg

/

μl

H2O distilled

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3. Electrophoresis

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3.1 Open the gel cassette and

moisten the pockets with

distilled water.

3.2 Excess water is

removed.

4 groups are working together in one gel cassette.

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3. Electrophoresis

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3.3 Pipette 4

μl

loading buffer to your samples (K, 1, 2).

3.4 Pipette into the pockets of the

gel 5

μl

of

-

kontrol

(K),

- samples 1 and 2

and once 5

μl

marker (M) in

the middle pocket of the gel

3.5 Start the electrophoresis

at 180 V.

CpG

methylation and ClaI restrictionAgarose gel electrophoresis - overview

Flash-Gel: 1,2 % 12+1, 180 Volt

GR 1

GR 2

GR 3

GR 4

Gel

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M = Lambda-DNA /EcoRI plus

HindIII

Marker,

ready-to-use

K 1 2 K 1 2 M K 1 2 K 1 2

Restriction

of

CpG-methylated and non-methylated λ-DNA with ClaI

CpG-methylated5‘ … ACAACATCGAT

AATGAATCAT … 3‘

Sequence for ClaIto recognize

and cut

Restriction

enzyme

Cla

I

Methyl

group

on

CpG

5‘ … ACAAC

AT

Sequence

for

Cla

I

to

recognize

and

cut

CGAT

AATGAATCAT … 3‘

Restriction

enzyme

Cla

I

CpG-unmethylated

Result of the methylation experiment

M

K1 (CpG-)P1 (CpG- +ClaI

P2 (CpG++ClaIP1 (CpG

-. ClaI)P2 (CpG+. ClaI)

-

+

Experimental

conditions

: Methylation reaction 90 min.; 37 °C Inactivation: 20 min., 65 °C;

ClaI-restriction: 30 min., 37 °C