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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
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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
Nucleic acids gel electrophoresis
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.
Nucleic acids gel electrophoresis
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.
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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
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Electrophoresis on agarose: NA visualization
Hoechst 33258
Electrophoresis on agarose: NA visualization
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
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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
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Electrophoresis on agarose: examples
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
Electrophoresis on agarose: examples
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
Electrophoresis on agarose: examples
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 _______________ _______________ _______________ ________________++ --
Electrophoresis on agarose: examples
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
Electrophoresis on agarose: examples
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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
Electrophoresis on agarose: examples
II
I
ri
+ distamycin- distamycin
0.5 1.0 3.0 control0.5 1.0 3.0 control
[PdCl2L5] Does not bindin minorgroove
Electrophoresis on agarose: examples
[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
Electrophoresis on agarose: examples
[Pd(L6)2]
Electrophoresis on agarose: examples