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 Restricciรณn
Extremos cohesivos
Extremos romos
Clonaciรณn 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 dNTP’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 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
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 (intragÊnicos)  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 restricciรณn
VNTRs o microsatélites Pueden hacerse por Southern blot Por PCR y geles de poliacrilamida o agarosa Por SSCP
Cepas diferentes de Salmonellas