adn cyted2 - riidfcm-cyted.fq.edu.uy · metálicos con adn: iv. electroforesis en gel prof. dr....

24/07/2010

1

Técnicas de estudio de la interacción de compuestos

metálicos con ADN:

IV. Electroforesis en gel

Prof. Dr. Dinorah GambinoCátedra de Química Inorgánica, Facultad de Química,

Universidad de la RepúblicaMontevideo, Uruguay

1. Basics of electrophoresis1. Basics of electrophoresis

2. Nucleic acids gel electrophoresis2. Nucleic acids gel electrophoresis

3. Agarose gel electrophoresis3. Agarose gel electrophoresis

4. Examples4. Examples

Gel electrophoresis

most biological polymers are charged and will

move in an electric field

characteriza-tion of a

molecule by its rate of

movement in the electric

field

determine protein MW

distinguish molecules by their net charge or shape

separate molecular species

quantitatively

Transport of particles through a solvent by an

electric field

Transport of particles through a solvent by an

electric field

Electrophoresis

Electrophoresis

molecule of charge qsubmitted to an electric field E

Eq ==== fv

µ ==== v////E ==== q////f

electrical force viscous drag

f frictional coefficientµ mobility

Gel electrophoresis

separation of charged molecules through

a matrix

matrix: paper, starch, agarose, polyacrylamide

24/07/2010

2

Gel electrophoresis: parameters

matrix composi-tion

parameters

strength of E

buffer composi-tion

molecules nature

sizeshapecharge

chemical composition

Nucleic acids gel electrophoresis

polinucleotides:charge////mass ≅≅≅≅ k

Migration of nucleic acids is inverselyproportional to size.

Nucleic acids gel electrophoresismigration

size

12 kb

4kb

1kb

agarose gel with a 1 kb ladder, right lane


A DNA ladder is a solution of DNA molecules of different

lengths used as a reference to estimate the size of unknown

DNA molecules.

Nucleic acids are separated on a gel or matrix of agarose oracrylamide.

The gel is prepared in a mold withwells to put the sample.

Each end of the gel is in contactwith the electrophoresis buffer orthe whole gel is immersed in it.

Buffer ions transport the chargeand mantain the pH valueapproximately constant.


Agarose is a polysaccharideobtained from marine algae.

Electrophoresis on agarose gels: agarose

Low melting point(65ºC) agarosebeds are used in Molecular Biologyexperiments for theanalysis and separation of DNA fragments and shapes. It allowsbands isolation and quantification.

24/07/2010

3

Electrophoresis on agarose: % of agarose

size range

5-10kb

0.2-1kb

very smallfragments

% agarose

0.7

2.0

3.0

% agarose 0.7-2.0 in buffer (TBE or TAE)

resolution varies with % agarose

Electrophoresis on agarose gels: technique

Gel electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical field is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera.

Electrophoresis on agarose gels: sample

buffer

dye*

MW marker

well size, number

volume: 10-20 µL

* bromophenol blue (300bp), xylencianol (4kb)

Electrophoresis on agarose: intensity

generally: 50-100 V for a couple of hours

High voltagesdiminish resolution.

fluorescent intercalator: absorbs light andemits in the visible region (orange); itsquantum yield is enhanced when bound toDNA

included in the gel before run, added tothe sample in the buffer or afterelectrophoretic run

a transiluminator is needed

Electrophoresis on agarose: NA visualization

stain: ethidiumbromide

24/07/2010

4


Hoechst 33258


ethidium bromide: carcinogenic, mutagenic

Electrophoresis on agarose: NA quantification Electrophoresis on agarose: plasmid DNA

form Isupercoiled

form IIrelaxedcircular

form IIIlinear

Electrophoresis on agarose: plasmid DNA Electrophoresis on agarose: plasmid DNA

IIIII

I

24/07/2010

5

Electrophoresis on agarose: plasmid DNA

intercalation

intrastrand

cleveage

outer-sphere

interstrand

Electrophoresis on agarose: interaction

Neglected parasitic diseases

Schistosomiasis

Leishmaniasis

Trypanosomiasis

Malaria

vector:Triatoma infestans

parasite:Trypanosoma cruzi

Chagas disease: American Trypanosomiasis

Bioactive metal complexes: Mechanism of action

M-bioactive ligandM-bioactive ligand

2 parasitic targets: dual mechanism of action

* * ** # # # # ##

10G ROS

Electrophoresis on agarose: examples

Plasmid DNA (pEGFP (Clontech) or pBSK II BlueScript (Stratagene)

[RuIICl2(DMSO)2L]

NNH

O

NHROO2N

n

n = 0,1 R = H RuS1, RuS3n = 0,1 R = butyl RuS2, RuS4

RuCl

Cl

DMSO

DMSO

24/07/2010

6


RuS1control1 2 RuS2 RuS4RuS3

III

ri -- ---

0.1 0.5 1 2 4 0.10.51 2 4 0.1 0.5 1 2 4 0.10.5 1 2 4

ri = mol of complex: mol of DNA base pair

37 ºC, 24 h incubation

0 1 2 3 40.981.001.021.041.061.081.101.121.141.161.181.20

1/ R

elat

ive

mob

ility

ri0 1 2 3 4

50

55

60

65

70

75

80

85

% R

elax

ed c

ircu

lar

ri ▪: RuS1 ●: RuS2 ▲: RuS3

RuS3


I

II

I

II

ri C 0.1 1.0 4.0 C 0.1 1.0 4.0

37ºC 50ºC

EtBradded

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0

10

20

30

40

50

60

70

80

90

% S

uper

coile

d

Time (h)

RuS3


ri= 4

Time course

B

0 1 2 3 4 5

0.76

0.77

0.78

0.79

0.80

0.81

0.82

Rf

Tiempo (h)

I

II

III

time (h) 0 0.5 1 1.5 2 3 5

A RuS3Time course

- distamycin + distamycinri

1 2 3 4 5 6 7 8 9 10 11 12

I

II

DMSO _______________ _______________ _______________ ________________++ --


N

NH

S

NHRO

O2N

n

n = 0,1 R = H L1, L5n = 0,1 R = metil L2, L6n = 0,1 R = etil L3, L7n = 0,1 R = fenil L4, L8

N

N

S

NHRO

O2N

N

N

S

RHN

M

[MII(L-H)2]

n

n

ONO2

N

NH

S

NHROO2N

Cl

ClM

[MIICl2(L)]

n

2 series:

MN

NS

S

MS

ClN

Cl


24/07/2010

7

II

I

- 0.1 0.25 0.5 1.0 3.0 6.0 ri

[PdCl2L6]

ri = mol complex/mol base pairs

dose-dependentdiminution of

supercoiled DNA mobility


II

I

ri

+ distamycin- distamycin

0.5 1.0 3.0 control0.5 1.0 3.0 control

[PdCl2L5] Does not bindin minorgroove


[PdCl2L7] [PdCl2L6]

[PdCl2L]complexes

show a quick effect on DNA

II

I

0 1 2 3 4 570

75

80

85

90

95

100 [PdCl2(L6)]

[PdCl2(L7)]

migration/% (respect to control)

time/h

time (h) 0 0.25 0.5 1.0 2.0 3.0 4.0 5.0 0 0.25 0.5 1.0 2.0 3.0 4.0 5.0


[Pd(L6)2]


adn cyted2 - riidfcm-cyted.fq.edu.uy · metálicos con adn: iv. electroforesis en gel prof. dr....

Documents