06-dna recombinante.pdf

7/28/2019 06-DNA recombinante.pdf

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06. Tcnicas de DNA recombinante


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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


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Restriction enzymes

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


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Restriction enzymes

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


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Separao eletroforetica de grandes molculas de DNAPulse field eletrophoresis


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Separation of yeast chromosomes


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Caratersticas essenciais:

- Capacidade de incorporar DNA exgeno- Capacidade de replicao autnoma- Locais nicos de corte por ER- Mecanismo de seleo (resistncia antibitico)-

Fcil purificao


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Plasmideo pBR322Seleo por resistncia a antibiticos Amp ou Tet


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Polylinker or Multiple Cloning Site (MCS)


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Seleo por B-galactosidase


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Blue-white plasmid selection

B-gal +

B-Gal -


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Promotores T7 e T3 no polylinker


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CsCl2 Plasmid purification


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Electroporation


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Electroporation

GFP

Ph DNA t


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Phage DNA as vectors

L bd h lif l


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Lambda phage life cycles

Lysogeny


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Phage DNA replication and packaging


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Lambda vectors - insertion


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Lambda vectors - substitution


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Cosmid library preparation

Cosmid vectors


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Cosmid vectors

B t i l tifi i l h (BAC )


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Bacterial artificial chromosomes (BACs)


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Yeast artificial chromosome vector (YACs)


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Expression of recombinant proteins

Fusion proteins


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T7 polymerase activation of plasmid transcription

His-tagged fusion proteins


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His tagged fusion proteins


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Purification of HIS-tagged fusion proteins

DNA lib i


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DNA libraries


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BibliotecaGenomica

Cosmideo

Biblioteca Genomica BAC Digesto parcial


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Biblioteca Genomica BAC

Sau3A 5GATC3

BamHI 5GGATCC3


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Non radioactive DNA labelling


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Non-radioactive DNA labelling


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Labelling DNA ends


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Labelling DNA ends

Chromosome walking


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Chromosome walking

cDNA libraries


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Isolamento de mRNA utilizandooligo dT e magnetic beads

mRNA - clula especfica

- tecido especfico

- estdio de desenvolvimento- condio especfica

c b a es


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Genomic e cDNA libraries


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Ratreiro de uma biblioteca de expresso


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Sequenciao do DNA

1. Mtodo qumico Maxam and Gilbert2. Mtodo enzimtico - Sangres- 500-800 pb max- Sequencia 1 cadeia de cada vez- DNA cadeia simples


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DNA sequencing gels


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DNA sequencing gels


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RandomShotguncloning


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DNAdeumBac-recombinante

Digestoparcial

ClonagemnumplasmideoemE.coli

123456789101112131415

123456789101112131415

123456789101112131415(n)

12345678789101112

91011121314

345678910567891011

1011121314

789101112

345678910

1011121314

1011121314

BibliotecadoDNAdoBac

Clonesindividuais

SequenciaoautomcaDNA


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Sequenciao automca NA

F

R

Fragmento



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q



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q



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q

Sequenciadorautomcoporcapilar

Resultadosobdosnocomputador

R t d G


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ReconstruodoGenoma

1345

5678

78910

101112

121314

111213

1345 91011

181920

202122

222324

212223

13456789101112131415161718192021222324

14151617

15161718

Sequnciadosclones

Individuais

Sequnciafinal


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Identification of the ORF


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Next generation sequencing - NGS


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Solid DNA sequencing:


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Hundreds of genomes sequenced so far:


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Northern Blot


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DNA microarrays

Arrayer


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

Amplificao por PCR

Printing (glass slide)

...........

...........

...........

...........

Cada spot um DNA deum gene diferente

Inkjet technology 10000 spots/cm2

DNA chips

DNA microarrays

Experincia:


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Experincia:

Clulas a crescer na condio

1 2

Gene expresso em 1

Gene expresso em 1 e2

Gene no espresso em 1 e2

Gene expresso em 2

Scanner

DNA microarrays: Computer analysis


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DNA microarrays: cluster analysis


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DNA microarrays: cluster analysis


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Ordenar os resultados do microarray em funo de alguma varivel

Experincia: fibroblastos em cultura

retirar soro fetal durante 48 horasadicionar 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 expressoPreto = sem alterao

Expression of different genes in different cells types


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DNA micro-arrays

Genes 12

(n)

RNA sequencing

Bar codes 4 12 n


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Bar codes 4-12 n

Polymerase chain reaction (PCR)


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DNA footprint


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The Nobel Prize in Physiology or Medicine 2006 was awarded jointlyto Andrew Z. Fire and Craig C. Mello "for their discovery of RNA

interference - gene silencing by double-stranded RNA"

Andrew Z. Fire Craig C. Mello

The Nobel Prize in Physiology or Medicine 2006


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Prevent translationPromote mRNA degradation

RNA-induced silencing complex (RISC)


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Endogenous micro RNAs control many aspects of gene expression


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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


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a d e ted d seases

A mutation in the seed region of miR-96 causes hereditary progressivehearing loss.A mutation in the seed region of miR-184 causes hereditary

keratoconus with anterior polar cataract. Deletion of the miR-17~92 clustercauses 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 foundto 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


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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 ofembryonic stem cells".

Photos: Copyright The Nobel Foundation

y gy


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