06.

Tcnicas de DNA recombinante

Restriction enzymes
- - - Reconhecem sequencias especificas 4-6-8-10-12 pb Designadas em relao a bactria ondo foi isolada Ao cortar o DNA podem gerar extremidades 3 ou 5

Restriction enzymes
DNA molecules treated with REs giving 3 or 5 overhang can be put back again

Restriction enzymes
DNA molecules treated with REs giving no overhang can also be put back again but a low frequency

Separao eletroforetica de grandes molculas de DNA Pulse field eletrophoresis

Separation of yeast chromosomes

Caratersticas essenciais: - - - - - Capacidade de incorporar DNA exgeno Capacidade de replicao autnoma Locais nicos de corte por ER Mecanismo de seleo (resistncia antibitico) Fcil purificao

Plasmideo pBR322 Seleo por resistncia a antibiticos Amp ou Tet

Polylinker or Multiple Cloning Site (MCS)

Seleo por B-galactosidase

Blue-white plasmid selection

B-gal + B-Gal -

Promotores T7 e T3 no polylinker

CsCl2 Plasmid purification

CsCl2 Plasmid purification

CsCl2 Plasmid purification

Electroporation

Electroporation

GFP

Phage DNA as vectors

Lambda phage life cycles

Lysogeny

Phage DNA replication and packaging

Lambda vectors - insertion

Lambda vectors - substitution

Cosmid library preparation

Cosmid vectors

Bacterial artificial chromosomes (BACs)

Yeast artificial chromosome vector (YACs)

Expression of recombinant proteins

Fusion proteins

T7 polymerase activation of plasmid transcription

His-tagged fusion proteins

Purification of HIS-tagged fusion proteins

DNA libraries

Biblioteca Genomica Cosmideo

Biblioteca Genomica BAC

Digesto parcial Sau3A 5GATC3 BamHI 5GGATCC3

Non-radioactive DNA labelling

Labelling DNA ends

Chromosome walking

cDNA libraries

Isolamento de mRNA utilizando oligo dT e magnetic beads mRNA - clula especfica - tecido especfico - estdio de desenvolvimento - condio especfica

Genomic e cDNA libraries

Ratreiro de uma biblioteca de expresso

Sequenciao do DNA 1. Mtodo qumico Maxam and Gilbert 2. Mtodo enzimtico - Sangres - 500-800 pb max - Sequencia 1 cadeia de cada vez - DNA cadeia simples

DNA sequencing gels

Random Shotgun cloning Biblioteca do DNA do Bac DNA de um Bac-recombinante

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (n) 3 4 5 6 7 8 9 10 7 8 9 10 11 12

Digesto parcial
1 2 3 4 5 6 7 8 9 10 11 12 13 14 5 6 7 8 9 10 11 3 4 5 6 7 8 9 10 7 8 9 10 11 12

10 11 12 13 14

10 11 12 13 14

10 11 12 13 14

Clones individuais

Clonagem num plasmideo em E.coli

Sequenciao automNca DNA

Fragmento

Sequenciao automNca DNA

Sequenciao automNca DNA

Sequenciao automNca DNA

Resultados obNdos no computador

Sequenciador automNco por capilar

Reconstruo do Genoma

Sequncia dos clones Individuais Sequncia nal

1 3 4 5

7 8 9 10

12 13 14 14 15 16 17

18 19 20 22 23 24 20 21 22 21 22 23

5 6 7 8 1 3 4 5

10 11 12 9 10 11 11 12 13

15 16 17 18

1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Identification of the ORF

Next generation sequencing - NGS

Solid DNA sequencing:

Hundreds of genomes sequenced so far:

Northern Blot

DNA microarrays
Coleco de DNAs especficos para cada gene (cDNAs, ESTs, fragmentos)

Arrayer

Amplificao por PCR

Printing (glass slide)

DNA chips

Cada spot um DNA de um gene diferente

........... ........... ........... ...........

Inkjet technology

10000 spots/cm2

DNA microarrays Experincia:

Clulas a crescer na condio 1 2

Scanner

Gene expresso em 1 Gene expresso em 1 e2

Gene no espresso em 1 e 2 Gene expresso em 2

DNA microarrays: Computer analysis

DNA microarrays: cluster analysis

DNA microarrays: cluster analysis

Ordenar os resultados do microarray em funo de alguma varivel Experincia: fibroblastos em cultura retirar soro fetal durante 48 horas adicionar soro ao tempo 0 e recolher amostra de referncia (marcar verde) recolher amostras de cDNAs nos diferentes tempos (marcar vermelho) comparar com uma amostra de referncia (hibridar cada tempo com amostra de referncia) aplicar mistura ao microarray Vermelho = aumento da expresso Verde = reduo da expresso Preto = sem alterao

Expression of different genes in different cells types

Genes 1 2 (n)

DNA micro-arrays

RNA sequencing Bar codes 4-12 n

Polymerase chain reaction (PCR)

DNA footprint

The Nobel Prize in Physiology or Medicine 2006

Andrew Z. Fire

Craig C. Mello

The Nobel Prize in Physiology or Medicine 2006 was awarded jointly to Andrew Z. Fire and Craig C. Mello "for their discovery of RNA interference - gene silencing by double-stranded RNA"

Prevent translation Promote mRNA degradation

RNA-induced silencing complex (RISC)

Endogenous micro RNAs control many aspects of gene expression

22 n non coding Plants perfect pairing Animals impefect pairing Human genome: 1000 microRNAs Human target: 60% genes miR= RNA polimerase II Intergenic, near host genes 48% miRs polycistronic units Promoters similar to genes miR: poly (A), CAP 5 end pre-miRNA: up to 6 miRNA precursors RNA editing

miRNA and inherited diseases A mutation in the seed region of miR-96 causes hereditary progressive hearing loss.A mutation in the seed region of miR-184 causes hereditary keratoconus with anterior polar cataract. Deletion of the miR-17~92 cluster causes skeletal and growth defects. miRNA and cancer The first human disease known to be associated with miRNA deregulation was chronic lymphocytic leukemia and later many miRNAs have been found to have links with some types of cancer. miRs: regulation of virtually all aspects of mammalian gene expression.

The Nobel Prize in Physiology or Medicine 2007

Mario R. Capecchi

Sir Martin J. Evans

Oliver Smithies

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells". Photos: Copyright The Nobel Foundation