TECNICAS DE BIOLOGНA MOLECULAR

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TECNICAS DE BIOLOGÍA MOLECULAR
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ENDONUCLEASASO ENZIMAS DE RESTRICCIÓN

• Generan cortes internos en la molécula de ADN
• Pueden generar extremos romos o cohesivos

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Enzimas de Restricción
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Extremos cohesivos
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Extremos romos
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Clonación y uso de enzimas
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Separación de fragmentos

• Geles de Agarosa
• Geles de poliacrilamida

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CLONACIÓN
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PCR (reacción en cadena de polimerasa)
Componentes de la reacción

• Polimerasa
• Primers
• dNTPs
• Cloruro de magnesio
• Agua
• Buffer

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SOUTHERN BLOT
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1) Blood sample. 2) DNA is extracted from blood
cells. 3) DNA is cut into fragments by a
restriction enzyme. 4) The DNA fragments are
separated into bands during electrophoresis in
an agarose gel. 5) The DNA band pattern in the
gel is transferred to a nylon membrane. 6)
Radioactive DNA probe, designed to recognise
specific DNA sequences, is prepared. 7) Probe
DNA binds to the specific DNA sequences on the
membrane. 8) Excess DNA is washed off, but the
DNA pattern is invisible at this stage. 9)
X-ray film is placed next to the membrane amp
exposed to the radioactive DNA. 10) The
developed X-ray shows the pattern of DNA bands
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SECUENCIACIÓN
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VNTR
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DNA fingerprinting in Argentina to establish
kinship of the 'disappeared', the children, often
born in captivity, of parents kidnapped and
assassinated by the former military regime there.
Here a geneticist uses a multi-channel pipette to
prepare samples for the process. A bank of blood
samples, taken from all the potential relatives,
provides the DNA for comparison with that from
the child in question. Fragments of DNA are
seperated into bands by electrophoresis in an
agarose gel. Every human being produces a unique
pattern of DNA bands, hence its potential as a
kind of genetic
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CLONACIÓN EN ANIMALES
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COPY-CAT (EE.UU 2002)
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PAMPA (Argentina 2002)
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PROMETEA (Italia 2003)
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Ian Wilmut and Dolly. In 1996, British
embryologist Professor Ian Wilmut created, the
world's first sheep cloned from an adult sheep
cell. The research was conducted at the Roslin
Institute in Edinburgh, Scotland.
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Marcadores moleculares

• Y sus aplicaciones

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Tipos de Marcadores

• Directos o sea dentro del gen (intragénicos)
• Indirectos usando a otro gen o marcador cercano
  en el cromosoma para poder identificar sus
  diferencias entre individuos

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RFLPs

• Pueden hacerse por Southern blot
• Pueden hacerse por PCR

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Utilidad

• Detección de individuos portadores de un gen
  recesivo.
• Test de paternidad o trazabilidad.
• Selección asistida por marcadores,
• Salud publica,
• Virología, etc

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Por Southern blot
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Por PCR y luego restricción
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VNTRs o microsatélites

• Pueden hacerse por Southern blot
• Por PCR y geles de poliacrilamida o agarosa
• Por SSCP

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Cepas diferentes de Salmonellas
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