TECNICAS DE BIOLOGA MOLECULAR ENDONUCLEASAS O ENZIMAS DE

TECNICAS DE BIOLOGÍA MOLECULAR

ENDONUCLEASAS O ENZIMAS DE RESTRICCIÓN § Generan cortes internos en la molécula de ADN § Pueden generar extremos romos o cohesivos

Enzimas de Restricción

Extremos cohesivos

Extremos romos

Clonación y uso de enzimas

Separación de fragmentos § Geles de Agarosa § Geles de poliacrilamida

CLONACIÓN

PCR (reacción en cadena de polimerasa) Componentes de la reacción § Polimerasa § Primers § d. NTP’s § Cloruro de magnesio § Agua § Buffer

SOUTHERN BLOT

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 & exposed to the radioactive DNA. 10) The developed X-ray shows the pattern of DNA bands

SECUENCIACIÓN

VNTR

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 multichannel 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

CLONACIÓN EN ANIMALES

COPY-CAT (EE. UU 2002)

PAMPA (Argentina 2002)

PROMETEA (Italia 2003)

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.

Marcadores moleculares Y sus aplicaciones

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

RFLP’s § Pueden hacerse por Southern blot § Pueden hacerse por PCR

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

Por Southern blot

Por PCR y luego restricción

VNTRs o microsatélites § Pueden hacerse por Southern blot § Por PCR y geles de poliacrilamida o agarosa § Por SSCP

Cepas diferentes de Salmonellas