Electroforesis

TODOS LO HACEN




Radio de giro vs.
tamao de poro
Flexibilidad vs.
tamao
,
Donde R es el radio de giro del polmero, N es el nmero de
segmentos de enlace (igual al grado de polimerizacin) de la cadena.

Para un buen disolvente, v = 3/5 ;
para un mal disolvente, v = 1/3 .
Por tanto un polmero en buen disolvente tiene un tamao mayor. En un
mal disolvente se comporta como una esfera slida.
Dependencia
N. C. Stellwagen and E. Stellwagen, Effect of the matrix on
DNA electrophoretic mobility, Journal of Chromatography A,
vol. 1216, no. 10, pp. 1917-1929, Mar. 2009.
Cuando el radio de giro de la molcula es
menor al tamao promedio de los poros.
0.5,
1.6,
3.1,
6.1,
9.2,
12.2 kbp
Dependencia
0.5,
1.6,
3.1,
6.1,
9.2,
12.2 kbp
N. C. Stellwagen and E. Stellwagen, Effect of the matrix on
DNA electrophoretic mobility, Journal of Chromatography A,
vol. 1216, no. 10, pp. 1917-1929, Mar. 2009.
Concentracin de
agarosa
En solucin
movilidad
Fraccin
disponible
del
volumen
En gel
Retardamiento
segn tamao
Cuando el radio de giro de la molcula es
menor al tamao promedio de los poros.
Dependencia
1.5%
1.25%,
0.9%,
0.6%,
0.4%,
0.2%,
N. C. Stellwagen and E. Stellwagen, Effect of the matrix on
DNA electrophoretic mobility, Journal of Chromatography A,
vol. 1216, no. 10, pp. 1917-1929, Mar. 2009.
Dependencia se pierde
D
i
s
t
a
n
c
i
a

r
e
c
o
r
r
i
d
a

e
n

u
n

t
i
e
m
p
o

t

Tamao en Kbp
Cuando el radio de giro de la molcula es
mayor al tamao promedio de los poros,
la molcula repta.
Reptar se divide en
Fase sin orientacin y
Fase con orientacin
for DNA chains approaching ~1020 kb the
electrophoretic mobility m becomes
increasingly independent of
molecular size
Elongated states form
because the molecules
remain hooked around gel
fibers, extending
both of the arms downfield,
here named U-shapes
The leading end is usually
bunched up, appearing
brighter than other parts of the
molecule, since it has to find
its
way through the gel pores
the molecules never stretch
totally to their theoretical
contour length. This is due to
the intrinsic entropic elasticity
of the molecules and also to
the zig-zag path through the
agarose network that they are
constrained to follow.
chain retardation is not
controlled by the time required
to form and slip out of the U-
shapes, but by the sieving of
the leading end through the
gel pores.
Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging of
the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees
and 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec
1;24(23):4759-4767.
Truco: campo pulsado
Se aplica un campo elctrico de ~10 V/cm,
o en ocasiones ms bajo, en forma
intermitente en dos direcciones.

La duracin ideal del pulso puede variar
desde menos de un segundo hasta varios
minutos, dependiendo del tamao de las
molculas a discriminar.
Se ha logrado la separacin de molculas
de hasta ~5 Mbp.
in DNA molecules undergoing 90 PFGE, the head of the
chain (that is bunched) usually remains the leading end of the
molecule and turns the corners in the direction of the new field,
entering new gel pores.
kinks (Fig. 4BC) (where
the molecule is doubled
over on itself) are
observed at the
beginning of the
reorientation, and these
disappear very rapidly.
leading end keeps driving the chain
both ends of the molecule
can extend and turn in the
new direction
Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging of
the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees
and 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec
1;24(23):4759-4767.
When the field is switched by an angle of 120 (or any other angle
.90o) the component of the new electric field along the previous
orientation of the molecule is now in a direction opposite to the
one along which the molecule was moving before and, therefore,
the molecules are pulled back along their previous paths.
Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging of
the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees
and 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec
1;24(23):4759-4767.
Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging of
the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees
and 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec
1;24(23):4759-4767.
Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging of
the reorientation mechanisms of YOYO-labelled DNA molecules during 90 degrees
and 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec
1;24(23):4759-4767.
Molculas individuales de
ADN
Caracterizacin mecnica con pinzas
pticas
Deposicin y comportamiento en superficie
observado con AFM
MICROSCOPIO DE PINZAS PTICAS

Tecnologa para estudiar
nanomquinas
Tecnologa para estudiar
nanomquinas
Manipulacin con pinzas lser
Principio de la deteccin de fuerza.








La aplicacin de una fuerza externa provocar un desplazamiento
fuera del centro de la trampa.
La desviacin del rayo difractado es medida por un fotodetector y
usada para calcular el valor de la fuerza aplicada sobre la
microesfera atrapada.
strep
anti-dig
Succin via
pipeta de vidrio
Trampa ptica
biotina
digoxigenina
DNA, 3-15 Kbp
strep
Succin via
pipeta de vidrio
Trampa ptica
DNA, 3-15 Kbp
anti-dig
strep
Succin via
pipeta de vidrio
Trampa
ptica
DNA, 3-15 Kbp
anti-dig
r
Lc
Modelo cadena vermiforme
(worm-like chain model)
Modelo cadena vermiforme
(worm-like chain model)
r
Lc
Does transition 3D2D affect
molecular conformation?
Free diffusion

Equilibrium is reached
Limited diffusion
(kinetical trap)

No equilibrium

Projection 3D
Analysis of the DNA end-to-end
distance distributions
8
fragmentos


pixel 4nm
(12pb)

DNA Fragment
(bp)
N
molecules
Contour Length
(nm)
Theoretical <R
2
>
(nm
2
)
Experimental <R
2
>
(nm
2
)
278 64 84 7 5533 4312 1235
731 101 218 16 26110 24727 11366
1450 110 404 32 63010 67332 37354
278bp 731bp 1450bp
Rivetti, 1996