Diapositiva 1

Plsmidosy csmidosAlejandro CardiniAL12517988Microbiologa y taxonoma microbiana

plsmidos20-100 copias/clulafragmento clonado: 5-10 Kpbtema 29 (13 pginas)2Vectores de clonacin o molculas transportadoras de DNA:mantienen y amplifican el fragmento clonado (se replican y aumentan en nmero)Caracteristicas:Pequeos y bien caracterizados Contienen: un origen de repli; sitios de cortes (RE) nicos; marcador (R a Abo gen que complementa a bacteria) Fciles de recuperar

fagos15-20 Kpb tema 29 (13 pginas)3Lambda genoma: 45 kpbPorcin central de su genoma, no es necesaria parasu replicacin (infeccin y formacin de calvas)

Figure: 17-08. Phage Lambda as a VectorPhage lambda as a vector. DNA is extracted from the phage, the central gene cluster is removed, and the DNA to be cloned is ligated into the arms of the phage lambda chromosome. The recombinant chromosome is then packaged into phage proteins to form a recombinant virus.

csmidosHbrido entre plsmido y fago:in vivo se mantiene como plsmido; in vitro se empaqueta como fago

Fragmento clonado: hasta 45 Kpb fagos de una cadena

Infeccin: 1 cadena; replicacin: 2 cadenas ( plsmidos)

tiles: secuenciacin y mutagnesis

Ej: el ms usado M13

BAC: cromosomas artificiales bacterianos

Basados en plsmido F (genes de replicacin y control de nmero de copias)

Vectores de expresin: sitios de restriccin flanqueados por promotores

tema 29 (13 pginas)4BAC: vectores para cartografiar y analizar genomas eucariticos complejosVectores de expresin vs vectores transbordadoresVector de expresin: seales de transcripcin y traduccinPlsmidos bifuncionales: con orgenes de replicacin de procariotas (pBR322) y eucariotas (SV40)

pBR322pUC18seleccin de plsmidos recombinantestema 29 (13 pginas)5seleccin de plsmidos recombinantes: RASTREO o CRIBADOpBR322: R a Ab en dos pasospUC18: X-gal un paso

seleccin del clon deseado (I)

tema 29 (13 pginas)6Suzuki, Fig. 12-05

seleccin del clon deseado (II)tema 29 (13 pginas)7Figure: 17-13a. Screening a Plasmid Library Illustrated procedure for screening a plasmid library to recover a cloned gene. Hybridization events are seen as spots on the film, and colonies containing the insert that hybridized to the probe are identified from the orientation of the spots; cells are selected from this colony for growth and further analysis.

seleccin del clon deseado (III)tema 29 (13 pginas)8Figure: 17-13b. Screening a Plasmid Library Illustrated procedure for screening a plasmid library to recover a cloned gene. Hybridization events are seen as spots on the film, and colonies containing the insert that hybridized to the probe are identified from the orientation of the spots; cells are selected from this colony for growth and further analysis.

VNTR: repeticiones en tndem de nmero variable Aplicaciones VNTR: - pruebas de paternidad - gentica forense - evolucin de poblacionestema 29 (13 pginas)9http://www.dnai.org/d/index.htmlVNTR un tipo de RFLP, pero no estrictamente lo mismo. Slo hace falta hacer un PCR! En lugar de Southern!# VNTR's are used in DNA fingerprinting: analisi forense y pruebas de paternidad# PCR has supplanted RFLP by Southern blotting and hybridization in analysis of VNTR's.Polymorphisms in the lengths of tandemly repeated short sequences can be used as molecular markers on the genetic chromosome.The term VNTR refers to the variable sequence rather than to the method used to detect it. In the description above, RFLP is used to detect the variability in the number of repeats. Several PCR-based methods can also be used to detect VNTR's. * Short identical segments of DNA aligned head to tail in a repeating fashion are interspersed in the human genome. * The numer of repeated segments at a locus varies between individuals. Such sequence regions are called VNTR's (variable number of tandem repeats). * Some VNTR sequence segments are found at only a single locus in the human genome. Probes made of these sequences are single locus probes and yield patterns such as those at left when used to probe RFLP blots of DNAs of six individuals. * Other VNTR sequence segments occur at many loci in the human genome. These loci are dispersed among the chromosomes. Multi-locus VNTR probes yield patterns such as those at right.

VNTR: pruebas de paternidadtema 29 (13 pginas)10VNTR un tipo de RFLP, pero no estrictamente lo mismo. Slo hace falta hacer un PCR! En lugar de Southern!DNA satlite: en ciertas zonas de varios cromosomas y en centrmeros y telmeros