2b -electroforesis moleculas individuales de dna -dg -bmc2014

8/11/2019 2B -Electroforesis Moleculas Individuales de DNA -DG -BMC2014

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Electroforesis

TODOS LO HACEN


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Radio de giro vs.

tamao de poro Flexibilidad vs.

tamao


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,

Donde R es el radio de girodel polmero, N es el nmero de

segmentos de enlace (igual al grado de polimerizacin) de la cadena.

Para un buendisolvente, v = 3/5 ;para un maldisolvente, v = 1/3 .Por tanto un polmero en buen disolvente tiene un tamao mayor. En unmal disolvente se comporta como una esfera slida.
http://es.wikipedia.org/wiki/Radio_de_giro_(ingenier%C3%ADa_estructural)http://es.wikipedia.org/wiki/Radio_de_giro_(ingenier%C3%ADa_estructural)http://es.wikipedia.org/wiki/Radio_de_giro_(ingenier%C3%ADa_estructural)


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Dependencia

N. C. Stellwagen and E. Stellwagen, Effect of the matrix onDNA electrophoretic mobility, Journal of Chromatography A,vol. 1216, no. 10, pp. 1917-1929, Mar. 2009.

Cuando el radio de giro de la molcula esmenor al tamao promedio de los poros.

0.5,1.6,3.1,6.1,9.2,12.2 kbp


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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 deagarosa

En solucin

movilidad

Fraccin

disponible

del

volumen

En gel Retardamientosegn tamao

Cuando el radio de giro de la molcula esmenor al tamao promedio de los poros.


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Dependencia

1.5%

1.25%,0.9%,0.6%,0.4%,0.2%,

N. C. Stellwagen and E. Stellwagen, Effect of the matrix onDNA electrophoretic mobility, Journal of Chromatography A,vol. 1216, no. 10, pp. 1917-1929, Mar. 2009.


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Dependencia se pierde

Distan

ciarecorrida

enun

tiempot

Tamao en Kbp

Cuando el radio de giro de la molcula esmayor al tamao promedio de los poros,la molcula repta.Reptar se divide enFase sin orientacin y

Fase con orientacin


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for DNA chains approaching ~1020 kb theelectrophoretic mobility m becomesincreasingly independent ofmolecular size


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Elongated states formbecause the moleculesremain hooked around gelfibers, extendingboth of the arms downfield,here named U-shapes

The leading end is usually

bunched up, appearingbrighter than other parts of themolecule, since it has to finditsway through the gel pores

the molecules never stretchtotally to their theoreticalcontour length. This is due tothe intrinsic entropic elasticityof the molecules and also tothe zig-zag path through theagarose network that they are

constrained to follow.

chain retardation is notcontrolled by the time requiredto form and slip out of the U-shapes, but by the sieving ofthe leading end through the

gel pores.

Gurrieri S, Smith SB, Wells KS, Johnson ID, Bustamante C. Real-time imaging ofthe reorientation mechanisms of YOYO-labelled DNA molecules during 90 degreesand 120 degrees pulsed field gel electrophoresis. Nucleic Acids Res. 1996 Dec1;24(23):4759-4767.


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Truco: campo pulsado

Se aplica un campo elctrico de ~10 V/cm,o en ocasiones ms bajo, en formaintermitente en dos direcciones.

La duracin ideal del pulso puede variardesde menos de un segundo hasta variosminutos, dependiendo del tamao de lasmolculas a discriminar.

Se ha logrado la separacin de molculasde hasta ~5 Mbp.


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in DNA molecules undergoing 90 PFGE, the head of the

chain (that is bunched) usually remains the leading end of themolecule and turns the corners in the direction of the new field,entering new gel pores.

kinks (Fig. 4BC) (wherethe molecule is doubledover on itself) areobserved at thebeginning of thereorientation, and thesedisappear very rapidly.

leading end keeps driving the chain

both ends of the moleculecan extend and turn in thenew direction



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When the field is switched by an angle of 120 (or any other angle.90o) the component of the new electric field along the previousorientation of the molecule is now in a direction opposite to theone along which the molecule was moving before and, therefore,the molecules are pulled back along their previous paths.



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Molculas individuales de

ADNCaracterizacin mecnica con pinzas

pticasDeposicin y comportamiento en superficie

observado con AFM


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MICROSCOPIO DE PINZAS PTICAS


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Tecnologa para estudiarnanomquinas


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Tecnologa para estudiarnanomquinas


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Manipulacin con pinzas lser Princip io de la deteccin de fuerza.

La aplicacin de una fuerza externa provocar un desplazamientofuera del centro de la trampa.

La desviacin del rayo difractado es medida por un fotodetector yusada para calcular el valor de la fuerza aplicada sobre lamicroesfera atrapada.


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strep anti-dig

Succin via

pipeta de vidrio Trampa ptica

biotinadigoxigenina

DNA, 3-15 Kbp


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strep

Succin via

pipeta de vidrio Trampa ptica

DNA, 3-15 Kbp

anti-dig


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strep

Succin viapipeta de vidrio

Trampaptica

DNA, 3-15 Kbp

anti-dig


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r

Lc

Modelo cadena vermiforme(worm-like chain model)


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Modelo cadena vermiforme(worm-like chain model)

r

Lc


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Does transition 3D2D affectmolecular conformation?

Free diffusion

Equilibrium is reached

Limited diffusion

(kinetical trap)

No equilibrium

Projection 3D


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Analysis of the DNA end-to-end

distance distributions

8fragmentos

pixel 4nm(12pb)


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DNA Fragment

(bp)

N

molecules

Contour Length

(nm)

Theoretical

(nm

2

)

Experimental

(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

2b -electroforesis moleculas individuales de dna -dg -bmc2014

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