Estratégias para isolamento de genes específicos de plantas

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Dr. Aulus Barbosa
Quais as ferramentas básicas 
para se isolar genes?
Endonucleases de Restrição
Transcriptase reversa
Eletroforese em gel
PCR
Vetores plasmidiais
Transformação de células
Purificação de plasmídeos
Bibliotecas de ácidos nucleicos
Endonucleases de restrição
• Cortam o DNA em sequências específicas
• Sistema imune de bactérias – 1950, 1962
• Primeira endonuclease isolada – 1970
 Haemophilus influenzae – HindII
 GTPyPuAC
• Tipos de endonucleases de restrição
 Tipo 1 – atividade de restrição e modificação
 Tipo 2 - atividade de restrição, cortam de maneira 
previsível ao lado do sítio de reconhecimento, 
necessitam somente de Mg++ como cofator.
 Tipo 3 - atividade de restrição e modificação
• Nucleases Tipo 2 
 Mais de 200 tipos conhecidos
 Reconhecem de 4 a 8 nucleotídeos
 Sequencias palindromicas
 http://highered.mcgraw-
hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction%20Endonucleases
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction Endonucleases
Enzyme Source
Recognition 
Sequence
Cut
EcoRI Escherichia coli 5'GAATTC 3'CTTAAG 
5'---G AATTC---3' 
3'---CTTAA G---5' 
BamHI
Bacillus
amyloliquefaciens
5'GGATCC 3'CCTAGG 
5'---G GATCC---3' 
3'---CCTAG G---5' 
HindIII
Haemophilus
influenzae
5'AAGCTT 3'TTCGAA 
5'---A AGCTT---3' 
3'---TTCGA A---5' 
TaqI Thermus aquaticus 5'TCGA 3'AGCT 
5'---T CGA---3' 
3'---AGC T---5' 
NotI Nocardia otitidis
5'GCGGCCGC 
3'CGCCGGCG 
5'---GC GGCCGC---
3' 3'---CGCCGG CG-
--5' 
HinfI
Haemophilus 
influenzae
5'GANTCA 3'CTNAGT 
5'---G ANTC---3' 
3'---CTNA G---5' 
Sau3A
Staphylococcus
aureus
5'GATC 3'CTAG 
5'--- GATC---3' 
3'---CTAG ---5' 
PovII* Proteus vulgaris 5'CAGCTG 3'GTCGAC 
5'---CAG CTG---3' 
3'---GTC GAC---5' 
SmaI*
Serratia
marcescens
5'CCCGGG 3'GGGCCC 
5'---CCC GGG---3' 
3'---GGG CCC---5' 
HaeIII*
Haemophilus
aegyptius
5'GGCC 3'CCGG 
5'---GG CC---3' 
3'---CC GG---5' 
HgaI[47]
Haemophilus
gallinarum
5'GACGC 3'CTGCG 
5'---NN NN---3' 
3'---NN NN---5' 
AluI*
Arthrobacter
luteus
5'AGCT 3'TCGA 
5'---AG CT---3' 
3'---TC GA---5'
http://en.wikipedia.org/wiki/EcoRI
http://en.wikipedia.org/wiki/Escherichia_coli
http://en.wikipedia.org/wiki/BamHI
http://en.wikipedia.org/wiki/Bacillus_amyloliquefaciens
http://en.wikipedia.org/wiki/HindIII
http://en.wikipedia.org/wiki/Haemophilus_influenzae
http://en.wikipedia.org/wiki/TaqI
http://en.wikipedia.org/wiki/Thermus_aquaticus
http://en.wikipedia.org/w/index.php?title=NotI&action=edit&redlink=1
http://en.wikipedia.org/w/index.php?title=Nocardia_otitidis&action=edit&redlink=1
http://en.wikipedia.org/w/index.php?title=HinfI&action=edit&redlink=1
http://en.wikipedia.org/wiki/Haemophilus_influenzae
http://en.wikipedia.org/w/index.php?title=Sau3A&action=edit&redlink=1
http://en.wikipedia.org/wiki/Staphylococcus_aureus
http://en.wikipedia.org/w/index.php?title=PovII&action=edit&redlink=1
http://en.wikipedia.org/wiki/Proteus_vulgaris
http://en.wikipedia.org/w/index.php?title=SmaI&action=edit&redlink=1
http://en.wikipedia.org/wiki/Serratia_marcescens
http://en.wikipedia.org/wiki/HaeIII
http://en.wikipedia.org/w/index.php?title=Haemophilus_aegyptius&action=edit&redlink=1
http://en.wikipedia.org/w/index.php?title=HgaI&action=edit&redlink=1
http://en.wikipedia.org/wiki/Restriction_enzyme
http://en.wikipedia.org/w/index.php?title=Haemophilus_gallinarum&action=edit&redlink=1
http://en.wikipedia.org/w/index.php?title=AluI&action=edit&redlink=1
http://en.wikipedia.org/w/index.php?title=Arthrobacter_luteus&action=edit&redlink=1
 Programas de computador podem ser usados para 
detectar sítios de restrição em sequências de DNA
 NEB Cutter
 http://tools.neb.com/NEBcutter2/
http://tools.neb.com/NEBcutter2/
Desenvolvida em 1983 por Kary Mullis
Rendeu o Prêmio Nobel de Química em 
1993.
Usava a DNA polimerase de Thermophilus
aquaticus – Taq Polimerase
Equipamentos necessários para PCR
• Termociclador
• Micropipetas
• Ponteiras
• Microtubos
• Freezer -20ºC
• Vortex
Reagentes para PCR
• Primers
• DNA molde
• Tampão
• dNTPs
• Taq-polimerase 
Como desenhar primers?
• Oligonucleotídeos sintéticos 
• Amplicons de 100 a 1000 pb
• Primer forward – início do gene
• Primer Reverse – fim do gene
• Os primers são escritos no sentido 5´- 3´
 Ex: Primers para amplificar o gene vif p23 de HIV
 Ferramenta para fazer o primer reverse
 http://www.vivo.colostate.edu/molkit/manip/index.html
http://www.vivo.colostate.edu/molkit/manip/index.html
>gi|145337680|ref|NM_106412.3| Arabidopsis thaliana ethylene-
responsive transcription factor ERF013 mRNA, complete cds
CTCATATTCCCAACACTTGTTCAAGCATATCTAAGTCAATAACAACATTACAACATAACATGGTG
AAACAAGAACTCAAGATCCAAGTTACTACTTCCTCATCATCACTCTCTCATTCTTCATCTTCTTC
TTCTTCTTCAACGTCGGCACTACGTCATCAATCTTGTAAGAACAAGATAAAGAAGTATAAAGGTG
TGAGGATGCGAAGTTGGGGATCATGGGTTACTGAAATTAGGGCACCAAATCAAAAGACAAGAATC
TGGTTAGGTTCTTACTCCACCGCAGAAGCAGCTGCTAGGGCTTACGACGCTGCTCTCTTGTGTCT
TAAGGGACCTAAAGCTAATCTCAACTTCCCTAACATCACCACTACTTCTCCTTTTCTTATGAACA
TCGACGAAAAGACCCTTTTGTCCCCAAAATCAATCCAAAAGGTCGCCGCTCAAGCCGCTAACTCC
TCTTCTGACCATTTTACCCCTCCGTCCGATGAAAATGATCATGATCATGATGACGGACTCGATCA
CCATCCATCTGCTTCTTCTTCAGCTGCATCTTCACCACCAGATGATGATCATCATAATGATGACG
ATGGTGATTTGGTATCGTTGATGGAATCTTTCGTGGATTACAACGAACACGTGTCTCTGATGGAT
CCATCGCTGTATGAATTTGGACATAATGAGATCTTCTTCACCAACGGAGATCCGTTTGATTATTC
TCCACAGTTACATAGCTCAGAGGCAACGATGGATGACTTCTACGACGATGTTGATATTCCGCTAT
GGAGTTTCAGTTAATCCAACGGTCCATATTATTATACTACGAAACACTTTTAATTCACATTATTT
CATTTAATTTATTCTTTCAATTCATATTATTCGTCCTTGTTGTTTCGTTATGTATTAAATATACA
CACACTAATTTAAGAAGATCACATGGACT
Primer Foward – CTCATATTCCCAACACTT
Primer Reverse - AGTCCATGTGATCTTCTT
Como saber se o primer está bom?
• Calcular a Tm – temperatura de anelamento
• Verificar a ocorrência de hairpins e Primer-Dimer
• Ferramenta – Oligo analyser
 http://www.idtdna.com/analyzer/applications/oligoanalyzer/
Hairpins Dimers
http://www.idtdna.com/analyzer/applications/oligoanalyzer/
 É possível desenhar primers sem ter a 
sequência?
• Usando sequências de organismos geneticamente 
próximos e procurando uma região conservada no 
gene de interesse
• Códigos para primers degenerados
R – A,C
Y – C,T
M – A,C
K – G,T
S – C,G
W – A,T
H – A,C,T
B – C,G,T
V – A,C,G
D – A,G,T
N – A,C,G,T
Como montar uma reação?
• 1 μL of 1 ng/μL lambda DNA (final amount = 1 ng).
• 1 μL of 50 μM forward PCR primer (final 
concentration = 1 μM).
• 1 μL of 50 μM reverse PCR primer (final 
concentration = 1 μM).
• 5 μL of 25 mM MgCl2 (final concentration = 2.5 mM).
• 4 μL of 2.5 mM dNTPs (final concentration = 200 
μM).
• 5 μL of 10× PCR buffer (final concentration = 1×).
• 0.25 μL of 5 U/μL Taq DNA polymerase (final amount 
= 1.25 U).
Como programar o termociclador?
• 95ºC – 5 min
• 95ºC – 1 min
• 45 – 60ºC – 30 seg Repetir 25 a 35 vezes
• 72ºC – 20 seg a 1 min
• 72ºC – 5 min
• 4ºC
Tipos de PCR
• PCR
• RT-PCR
 Usa a transcriptase reversa para amplificar mRNA
• RACE PCR - Rapid Amplification of cDNA Ends
 Liga adaptadores no cDNA dupla fita para amplificar todo
o gene de interesse
 5´ RACE PCR
 3´ RACE PCR
• NESTED PCR
 Utiliza duas PCRs em sequência, a segunda amplificando 
uma região interna a primeira PCR.
• Touchdown PCR
 Utiliza um programa que ao passar dos ciclos vai 
reduzindo a temperatura de anelamento da reação. 
 5´ RACE PCR
 3´ RACE PCR
 NESTED PCR
Eletroforese em Gel
• Desenvolvida em 1970 – Daniel Nathans
 Poliacrilamida
 Separava fragmentos de até 1000 pb
 DNA marcado radioativamente
• Joseph Sambrook – Cold Spring Harbor 
Laboratory - 1973
 Agarose
 Separava fragementos de 100 a mais de 50.000
 Coloração com brometo de etídio – detecta até 5 ng de 
DNA (0,000000005g)
http://www.biomolweb.kit.net/eletoforese.swf
Vetores plasmidiais
• Usados para carregar sequencias de DNA para 
uma célula hospedeira• Apresentam de 1000 a 200.000 pb
• DNA Circular
• Origem de replicação – ORI
• Genes de resistência a antibióticos
 Seleção das células transformadas
>pGEM-T Easy Vector, 3016 bp
GGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATnATCACTAGTGAATTCGCGGCCGCCTGCAGGTC
GACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTG
TTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGC
TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGG
AGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC
TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAA
AAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA
GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT
CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC
GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA
GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA
GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT
GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG
AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA
ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC
ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGC
TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT
ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC
TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC
TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA
TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA
ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG
CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA
GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT
CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCA
CCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTA
AATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGT
GTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTA
CGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGA
CGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGC
GTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGC
GGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT
GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATA
Como ligar genes em plasmídeos
• Enzimas de restrição
• DNA
• Plasmídeo
• DNA Ligase
Transformação de células
• Introdução do DNA em células hospedeiras
 Organismo hospedeiro
 Vetor com sequencias para replicação no organismo
 Seleção das células transformadas
• Escherichia coli
 Replicação a cada 22 minutos
 11 horas – 30 gerações – 1 bilhão de células
Transformação de células
• Transformação por choque térmico. 
 CaCl2 – Ca
++
 105 a 107 transformantes por micrograma de plasmídio
 Outros íons também podem ser usados – Mn++, K+ e 
Co++
 É provável que as células estejam mais competentes 
na fase log pois o DNA pode entrar através de poros 
nas zonas de adesão. 
 Dificilmente transforma DNAs maiores que 15 kb
Transformação de células
• Transformação por eletroporação
 Crescimento de células até fase log.
 Lavagem das bactérias com água pura ou deionizada.
 Fragmentos de DNA maiores podem ser eletroporados
– até 1000 kb. 
Extração dos plasmídeos recombinantes
• Miniprep, midiprep ou maxiprep?
 Miniprep – até 5 ml
 Midiprep – 15 a 25 ml
 Maxiprep – 100 a 200 ml
DNA Plasmid Miniprep Protocol
1. Pick single colony and inoculate 5 ml of LB broth containing 200 g/l ampicillin or 1mg/5ml. Optional: Use 
a 15ml conical tube with a loosened cap and a piece of tape to hold it in place. Shake at 250 RPM 37oC 
overnight.
2. Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1 minute. Aspirate supernatant.
3. Resuspend cell pellet in 100 ul of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 
8). Vortex gently if necessary.
4. Add 200 ul of NaOH/SDS lysis solution (0.2 M NaOH, 1% SDS). Invert tube 6-8 times.
5. IMMEDIATELY add 150 ul of 5 M potassium acetate solution (pH 4.8). This solution neutralizes NaOH in 
the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white, rubbery 
precipitate. Spin at top speed 1 min.
6. Transfer supernatant to new tube, being careful not to pick up any white flakes. Precipitate the nucleic 
acids with 0.5mL of isopropanol on ice for 10 minutes and centrifuge at top speed for 1 minute.
7. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 0.4 ml of TE buffer (10 mM Tris-Cl, 1 
mM EDTA, pH 7.5). Add 10ul of RNAse A solution (20 mg/ml stock stored at -20 °C), vortex and incubate 
at 37 °C for 20 to 30 minutes to digest remaining RNA.
8. Extract proteins from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol) by adding about 
0.3 ml. Vortex vigorously for 30 seconds. Centrifuge at full speed for 5 minutes at room temperature. 
Note organic PCIA layer will be at the bottom of the tube.
9. Remove upper aqueous layer containing the plasmid DNA carefully avoiding the white precipitated 
protein layer above the PCIA layer, transferring to a clean 1.5 ml epindorf tube.
10. Add 100 ml of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to precipitate the plasmid 
DNA on ice for 10 minutes. Centrifuge at full speed for 5 minutes at room temperature.
11. Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 50ul of DNA. Dissolve 5uL in 995ul 
of water, and spec (blank spectrophotometer to water). The absorbance at 260 nm multiplied by ten is 
the concentration of the DNA in units of mg/ml for a 1 cm pathlength cuvette (i.e. 50 mg/ml/OD 260nm).
http://sosnick.uchicago.edu/LB_broth.html
http://sosnick.uchicago.edu/GTE.html
http://sosnick.uchicago.edu/NaOH_SDS.html
http://sosnick.uchicago.edu/KOAc.html
http://sosnick.uchicago.edu/TE.html
 Seqüênciamento de DNA. 
 Seqüênciamento de DNA. 
 Seqüênciamento de DNA. 
 Seqüênciamento de ultima geração
• Roche/454 FLX 
• Illumina/Solexa Genome Analyzer 
• Applied Biosystems SOLiDTM System
• Helicos HeliscopeTM
• Pacific Biosciences SMRT
• Piroseqüênciamento
•Roche/454 FLX 
• Genomas completos
• Transcritoma
• Rápido e eficiente e em constante evolução
• 1.200.000 reads de 350 a 450 pb – 10 horas de corrida
• 500.000 reads de 100 a 250 pb – 10 horas de corrida
• Metodologia do Piroseqüênciamento (GS 454 
FLX Titanium)
• 3µg de cDNA - Shotgun method (remoção 
de polyA tails)
• Como funciona o Piroseqüênciamento?
• Preparação da amostra
• Adaptadores
• Single-strand bead binding
• emPCR
Sequenciamento - Ciclos GCTA
• Adaptador B – primer
• Ciclo 1 – dGTP → Polimerase libera PPi
• PPi + APS (Adenosine Phosphosulfate)
GCAGGCCT
ATP 
+
Luciferina
Sulfurilase
Oxi-luciferina
LuciferaseLuz
• Apirase
• Deoxyadenosine alfa-thio-triphosphate 
(dATP·αS) 
• Lavagem 
• Ciclo 2 – dCTP
• Cada emissão de luz em cada poço é registrada por ciclo de 
nucleotideo gerando uma foto da placa.
• Cadapoço corresponde a um read. Logo 1.200.000 “flowgrams” são 
gerados por cada corrida de 10 horas, sendo que as fotos em altíssima 
resolução são armazenadas imediatamente. 
• A quantidade de informação gerada por corrida é de cerca de 10 
Tbytes.