Desenho de Primers 2013

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Desenho de 
primers 
Universidade Federal do Rio Grande do Sul 
Instituto de Ciências Básicas da Saúde 
Laboratório de Virologia 
Equipe de Virologia – UFRGS & IPVDF 
 
www.ufrgs.br/labvir 
Primers 
• Sequencia de ácido nucleico que serve como 
um ponto de início para síntese de DNA 
• Na replicação de DNA a enzima que catalisa 
este processo somente pode adicionar 
nucleotídeos numa fita de DNA existente 
• A DNA polimerase inicia a replicação na 
extremidade 3’ do primer e copia a fita oposta 
Primers 
• Replicação do DNA in vivo 
ou DNA girase 
Proteínas SSP 
5` 
Primers 
• In vitro: quimicamente sintetizado para 
PCR e sequenciamento de DNA 
• Oligonucleotideos vão hibridizar com o 
DNA alvo 
• Determinar a sequencia de DNA que será 
amplificada no processo de PCR 
Uso de primers em Biologia Molecular 
• Clonar sequencias de DNA conhecidas 
 Uso de adaptadores, enzimas de restrição, etc 
• Clonar sequencias de DNA homólogas 
• Clonar sequencias de DNA não conhecidas 
• Sequenciamento de DNA 
• Uso de primers como sonda 
• Detecção de DNA (PCR) (90%) 
 
 
 
Clonar sequencias de DNA conhecidas 
• Por exemplo, a proteína Cap de PCV2 
codificada pela ORF2 
PCV2 
Rep Cap ori 
PF PR 
1776 pb 
ORF1 ORF2 
Clonar sequencias de DNA conhecidas 
• ORF 2 de PCV2 
aatacttacagcgcacttctttcgttttcagcgatgacgtatccaaggaggcgtttccgcagacgaagacaccgcccccg 
cagccatcttggccagatcctccgccgccgcccctggctcgtccacccccgccaccgttaccgctggagaaggaaaaatg 
gcatcttcaacacccgcctctcccgcaccatcggttatactgtcaagaaaaccacagtcagaacgccctcctggaatgtg 
gacatgatgagatttaatattaatgattttcttcccccaggagggggctcaaaccccctcactgtgccctttgaatacta 
cagaataaggaaggttaaggttgaattctggccctgctccccaatcacacagggtgacaggggagtgggctccactgctg 
ttattctagatgataactttgtaacaaaggccaatgccctaacctatgacccttatgtaaactactcctcccgccatacc 
ataacccagcccttctcctaccacccccggtactttaccccgaaacctgtccttgataggacaatcgattacttccaacc 
caataacaaaagaaatcaactctggctgagactacaaactactggaaatgtagaccatgtaggcctcggcactgcgtccg 
aaaacagtatatacgaccaggactacaatatccgtataaccatgtatgtacaattcagagaatttaatcttaaagacccc 
ccacttaaccctaagtgaataataaaa 
Forward primer 
Reverse primer 
Clonar sequencias de DNA conhecidas 
Região codificante da ORF 2 PCV2 
ATG Stop códon 
EcoRI 
Primer Forward 
Sequencia de Kozak 
Região do códon de início HindIII 
Primer Reverse 
Obs.: alterar a sequencia original para favorecer a expressão de proteínas 
Clonar sequencias de DNA conhecidas 
Sequencia de Kozak 
Clonar sequencias de DNA conhecidas 
ATG stop EcoRI 
Sequencia de Kozak 
HindIII 
EcoRI HindIII 
Promotor Vetor 
Região codificante da ORF 2 PCV2 
Clonar sequencias de DNA conhecidas 
Kampstrup et al., 2004 
Vaccine, 1358-1361 
Expressão de proteína Cap 
 de PCV2 em células PK15 
PCV2 
Rep Cap ori 
pF pR 
hCMV IE1 p. hCMV IE 5’ UT 
pcDNA3.1 
Cap 
Promotor 
ATGCAACCCACCGCGCCGCCCCGGCGGCGGTTGCTGCCGCTGCTGCTGCCGCAGTTATTGCTTTTCGGGCTGATGGCCGA
GGCCAAGCCCGCGACCGAAACCCCGGGCTCGGCTTCGGTCGACACGGTCTTCACGGCGCGCGCTGGCGCGCCCGTCTTTC
TCCCAGGGCCCGCGGCGCGCCCGGACGTGCGCGCCGTTCGCGGCTGGAGCGTCCTCGCGGGCGCCTGCTCGCCGCCCGTG
CCGGAGCCCGTCTGCCTCGACGACCGCGAGTGCTTCACCGACGTGGCCCTGGACGCGGCCTGCCTGCGAACCGCCCGCGT
GGCCCCGCTGGCCATCGCGGAGCTCGCCGAGCGGCCCGACTCAACGGGCGACAAAGAGTTTGTTCTCGCCGACCCGCACG
TCTCGGCGCAGCTGGGTCGCAACGCGACCGGGGTGCTGATCGCGGCCGCAGCCGAGGAGGACGGCGGCGTGTACTTCCTG
TACGACCGGCTCATCGGCGACGCCGGCGACGAGGAGACGCAGTTGGCGCTGACGCTGCAGGTCGCGACGGCCGGCGCGCA
GGGCGCCGCGCGGGACGAGGAGAGGGAACCAGCGACCGGGCCCACCCCCGGCCCGCCGCCCCACCGCACGACGACACGCG
CGCCCCCGCGGCGGCACGGCGCGCGCTTCCGCGTGCTGCCGTACCACTCCCACGTATACACCCCGGGCGATTCCTTTCTG
CTATCGGTGCGTCTGCAGTCTGAGTTTTTCGACGAGGCTCCCTTCTCGGCCAGCATCGACTGGTACTTCCTGCGGACGGC
CGGCGACTGCGCGCTCATCCGCATATACGAGACGTGCATCTTCCACCCCGAGGCACCGGCCTGCCTGCACCCCGCCGACG
CGCAGTGCAGCTTCGCGTCGCCGTACCGCTCCGAGACCGTGTACAGCCGGCTGTACGAGCAGTGCCGCCCGGACCCTGCC
GGTCGCTGGCCGCACGAGTGCGAGGGCGCCGCGTACGCGGCGCCCGTTGCGCACCTGCGTCCCGCCAATAACAGCGTAGA
CCTGGTCTTTGACGACGCGCCGGCTGCGGCCTCCGGGCTTTACGTCTTTGTGCTGCAGTACAACGGCCACGTGGAAGCTT
GGGACTACAGCCTAGTCGTTACTTCGGACCGTTTGGTGCGCGCGGTCACCGACCACACGCGCCCCGAGGCCGCAGCCGCC
GACGCTCCCGAGCCAGGCCCACCGCTCACCAGCGAGCCGGCGGGCGCGCCCACCGGGCCCGCGCCCTGGCTTGTGGTGCT
GGTGGGCGCGCTTGGACTCGCGGGACTGGTGGGCATCGCAGCCCTCGCCGTTCGGGTGTGCGCGCGCCGCGCAAGCCAGA
AGCGCACCTACGACATCCTCAACCCCTTCGGGCCCGTATACACCAGCTTGCCGACCAACGAGCCGCTCGACGTGGTGGTG
CCAGTTAGCGACGACGAATTTTCCCTCGACGAAGACTCTTTTGCGGATGACGACAGCGACGATGACGGGCCCGCTAGCAA
CCCCCCTGCGGATGCCTACGACCTCGCCGGCGCCCCAGAGCCAACTAGCGGGTTTGCGCGAGCCCCCGCCAACGGCACGC
GCTCGAGTCGCTCTGGGTTCAAAGTTTGGTTTAGGGACCCGCTTGAAGACGATGCCGCGCCAGCGCGGACCCCGGCCGCA
CCAGATTACACCGTGGTAGCAGCGCGACTCAAGTCCATCCTCCGCTAGGCGCCCCCCCCCCCGCGCGCTGTGCCGTCTGA
CGGAAAGCACCCGCGTGTAGGGCTGCATATAAA 
Primers para BoHV-1 (artigo Oliveira et al., 2013) 
PF: 5’-AAACCCCGCATATGGCTTCGGTCGACACGGTCTTCA-3’ 
gE 
(stop códon) 
NdeI: CA^TA_TG 
BamHI: G^GATC_C 
gE gene: 1793 pb 
gE RC: 1728 pb 
PCR: 651 pb 
Clonado em 
vector: pET16b 
PR: 5'-GTCGAAGGATCCAGACTGCAGACGCACCGATAG-3’ 
RC: 3’-CTATCGGTGCGTCTGCAGTCTGGATCCTTCGAC-5’ 
TATGGCTTCGGTCGACACGGTCTTCACGGCGCGCGCTGGCGCGCCCGTCTTTCTCCCAGGGCCCGCGGCGCGCCCGG
ACGTGCGCGCCGTTCGCGGCTGGAGCGTCCTCGCGGGCGCCTGCTCGCCGCCCGTGCCGGAGCCCGTCTGCCTCGAC
GACCGCGAGTGCTTCACCGACGTGGCCCTGGACGCGGCCTGCCTGCGAACCGCCCGCGTGGCCCCGCTGGCCATCGC
GGAGCTCGCCGAGCGGCCCGACTCAACGGGCGACAAAGAGTTTGTTCTCGCCGACCCGCACGTCTCGGCGCAGCTGG
GTCGCAACGCGACCGGGGTGCTGATCGCGGCCGCAGCCGAGGAGGACGGCGGCGTGTACTTCCTGTACGACCGGCTC
ATCGGCGACGCCGGCGACGAGGAGACGCAGTTGGCGCTGACGCTGCAGGTCGCGACGGCCGGCGCGCAGGGCGCCGC
GCGGGACGAGGAGAGGGAACCAGCGACCGGGCCCACCCCCGGCCCGCCGCCCCACCGCACGACGACACGCGCGCCCC
CGCGGCGGCACGGCGCGCGCTTCCGCGTGCTGCCGTACCACTCCCACGTATACACCCCGGGCGATTCCTTTCTGCTA
TCGGTGCGTCTGCAGTCTG 
Fragmento BoHV-1 (artigo Oliveira et al., 2013) 
gE 
Fragmento gE 
BoHV-1: 635 pb 
clonado em 
vector: pET16b 
NdeI: CA^TA_TG 
BamHI: G^GATC_C 
BoHV-1 Frame 2: 211 resíduos de aa 
 
MASVDTVFTARAGAPVFLPGPAARPDVRAVRGWSVLAGACSPPVPEPVCLDDRECFTDVALDA
ACLRTARVAPLAIAELAERPDSTGDKEFVLADPHVSAQLGRNATGVLIAAAAEEDGGVYFLYD
RLIGDAGDEETQLALTLQVATAGAQGAARDEEREPATGPTPGPPPHRTTTRAPPRRHGARFRV
LPYHSHVYTPGDSFLLSVRLQS 
ATGCGGGCCACCGCGCCGCCCCGGCCGCGGCTGCTGCCGCTGCTGCTGCCGCTGCTGCTGCCGCCGTCGCTCCTCGG
GCTACCGGTCGGGGCCGGCCTTGGCCCCAGCCCCAGCCCCGAAGCCGACACCGGGGCAAAGGCCCCGGCCGGCGCGG
TCTTCACCGCGCGCGTTGGTGCGCCCGTCTTCCTCCCTGGGCCTGACCCGCGCCCCGAGACGCGCGCCGTTCGCGGC
TGGAGCGTCCTCGCGAGCGACTGCCCACCGCCCGAGCCGACGCCCGTCTGCCTCGACGACCGCGAGTGCTTCGCCGA
CGTGGCCCTGGACGCGGCCTGCCTGCGGACCGCTCGCATGGCCCCGCTGGCCATCGCAGAGCTCACCGAGCGGCCCG
ATCCGGCGGGCGACAGGGAGTTCGTCGTCCCTGACCCGCGCGTTTCCGCGCGGCTGGGCCGCAACGCGACCGGGGTG
CAGATCGCGGACGTGACCGAGGAGGACGGCGGCGTGTACTTCCTGTACGACCGGGCCGCCGGCGACGCCGGCGACGA
GGAGACGCAGTCGACCCTGACGCTGCGGGTCGAGCCGGCCGACGCTTGGGACCCCGCCGGGCAGGGCGAGGGCGGGG
AAGGGGAAGGGGGGAAGGGGGGGCGAGGGGCGGCCAAGCCCACCCCCACCCCCACCCCCGCCCCCAGCCCGCCCCGC
CCCACGCCCGCGCGCCCCGCGCCCCCCCCGCGGCGGCGGCACGGCGCGCGCTTCCGCGTGCAGCCGTACCGCTCCCA
CGTGTACACCCCGGGCGACTCCTTCACGCTCTCGGTGCGGCTGCAGTCCGAGTTCTTCGACGAAGCGCCGTTCTCGG
CCAGCATCGACTGGTATTTTTTGCGGCCGGCCGGCGACTGCGCGCTCGTCCGCATCTACGAGACGTGCATCTTCCAC
CCCGAGGCGCCGGCCTGCCTGCACCCGGTCGACGCGCGGTGCGCCTTCGCGTCGCCCTACCGCTCCGAGACCGCGTA
CAGCCGGCTGTACGAGCGGTGCCGCCCAGCCTCCGCCGACCGCTGGCCGCGCGAGTGCGAGGGCGCTGCGTACGAGG
CCCCCGTCGCACACCTGCGCCCCGCCAACAACAGCGTGGACCTAGTCTTTGACGGCGCGCCGGCCTCGGCCTCGGGG
CTCTACGTCTTCGTGCTGCAGTACAACGGCCACGTGGAGGCCTGGGACTACAGCCTGGTCGTCACCTCGGACCGCCT
GGTGCGCGCCGTCACCGACCACACGCGCCCCGCGGCCGCCGACGCCCCCGAGCCGAGCCCGCCGCCCGCCGACGGGC
CGGCGGACGCGCCCGGCAGGGGCGCGCGCGGCCCCGCGCCCTGGCTCGTGGTGCTGGGGGGCGCGCTCGGGCTCGCG
GGCCTAATCGGCGTCGCGGCCCTCGCTGTCTGGGTGTGCGCGCGCCGCGCGGGCCAGAAGCGCACCTACGACATCCT
CAACCCCTTCGGGCCGGTGTACACCAGCCTGCCGACCAACGAGCCGCTCGACGTGGTGTCGGTCAGCGACGACGAGT
TCTTCCCCGACGAGGACTCTTCCTTCGCGGAGGACGGCAGCGACGCCGACGACGAGCTCGCCGACGAGCCCCCCGCCACCGCCGCCTACGACCTCGCCGGGCCCGCCCAGGGGGCCGGCGGGCCCGCGCGCCCGAGCCGCTCGGGCTTCAAGGT
CTGGTTTAGGGACCCGCTCGAGGACGACGATGTCGCGCCGGCGCGGCCCCAGACCGCGCCGGACTACACCGTGGTGG
CGGCGCGGCTGAAGTCCATCCTCC 
Primers para BoHV-5 (Siedler et al., in prep) 
PF: ACACCGGGGCAAAGGCCCCGGCCGGCGCGGTCTTCA 
gE 
GCTAG (stop códon original) 
BamHI: G^GATC_C 
KpnI: G_GTAC^C 
PR: 5'- GTCGAAGGTACCGGACTGCAGCCGCACCGAGAG-3’ 
PR: 3'-CTCTCGGTGCGGCTGCAGTCCGGTACCTTCGAC-5’ 
gE BoHV-5: 1795 pb 
PCR: 707 pb 
Clonado em 
vector: pAE 
GATCCGCCCCGGCCGGCGCGGTCTTCACCGCGCGCGTTGGTGCGCCCGTCTTCCTCCCTGGGCCTGACCCGCGCCCC
GAGACGCGCGCCGTTCGCGGCTGGAGCGTCCTCGCGAGCGACTGCCCACCGCCCGAGCCGACGCCCGTCTGCCTCGA
CGACCGCGAGTGCTTCGCCGACGTGGCCCTGGACGCGGCCTGCCTGCGGACCGCTCGCATGGCCCCGCTGGCCATCG
CAGAGCTCACCGAGCGGCCCGATCCGGCGGGCGACAGGGAGTTCGTCGTCCCTGACCCGCGCGTTTCCGCGCGGCTG
GGCCGCAACGCGACCGGGGTGCAGATCGCGGACGTGACCGAGGAGGACGGCGGCGTGTACTTCCTGTACGACCGGGC
CGCCGGCGACGCCGGCGACGAGGAGACGCAGTCGACCCTGACGCTGCGGGTCGAGCCGGCCGACGCTTGGGACCCCG
CCGGGCAGGGCGAGGGCGGGGAAGGGGAAGGGGGGAAGGGGGGGCGAGGGGCGGCCAAGCCCACCCCCACCCCCACC
CCCGCCCCCAGCCCGCCCCGCCCCACGCCCGCGCGCCCCGCGCCCCCCCCGCGGCGGCGGCACGGCGCGCGCTTCCG
CGTGCAGCCGTACCGCTCCCACGTGTACACCCCGGGCGACTCCTTCACGCTCTCGGTGCGGCTGCAGTCCG 
Fragmento BoHV-5 (Siedler et al., in prep) 
gE 
BamHI: G^GATC_C 
KpnI: G_GTAC^C 
Fragmento gE 
BoHV-5: 687 pb 
clonado em 
vector: pAE 
BoHV-5 Frame 3: 228 resíduos de aa 
 
HAPAGAVFTARVGAPVFLPGPDPRPETRAVRGWSVLASDCPPPEPTPVCLDDRECFADVALDA
ACLRTARMAPLAIAELTERPDPAGDREFVVPDPRVSARLGRNATGVQIADVTEEDGGVYFLYD
RAAGDAGDEETQSTLTLRVEPADAWDPAGQGEGGEGEGGKGGRGAAKPTPTPTPAPSPPRPTP
ARPAPPPRRRHGARFRVQPYRSHVYTPGDSFTLSVRLQS 
Comparação dos resíduos da gE de BoHV-1 (Oliveira 
et al., 2013) com a gE de BoHV-5 (Siedler et al., in 
prep) 
BoHV-5 Frame 3: 228 resíduos de aa 
 
HAPAGAVFTARVGAPVFLPGPDPRPETRAVRGWSVLASDCPPPEPTPVCLDDRECFADVALDA
ACLRTARMAPLAIAELTERPDPAGDREFVVPDPRVSARLGRNATGVQIADVTEEDGGVYFLYD
RAAGDAGDEETQSTLTLRVEPADAWDPAGQGEGGEGEGGKGGRGAAKPTPTPTPAPSPPRPTP
ARPAPPPRRRHGARFRVQPYRSHVYTPGDSFTLSVRLQS 
BoHV-1 Frame 2: 211 resíduos de aa 
 
MASVDTVFTARAGAPVFLPGPAARPDVRAVRGWSVLAGACSPPVPEPVCLDDRECFTDVALDA
ACLRTARVAPLAIAELAERPDSTGDKEFVLADPHVSAQLGRNATGVLIAAAAEEDGGVYFLYD
RLIGDAGDEETQLALTLQVATAGAQGAARDEE-------------REPATGPTPGPPPHRTT 
TR--APP--RRHGARFRVLPYHSHVYTPGDSFLLSVRLQS 
Primers para BoHV-5 (Siedler et al., in prep) 
Plasmídeo pAE 
Inserir o inserto relativo ao fragmento da gE de BoHV-5 nos sítios de 
BamHI: G^GATC_C 
KpnI: G_GTAC^C 
Primers para BoHV-5 (Siedler et al., in prep) 
Pretendemos utilizar o plasmídeo pAE (2822 pb) 
Inserir o inserto relativo ao fragmento da gE de BoHV-5 nos sítios de 
BamHI: G^GATC_C 
KpnI: G_GTAC^C 
gatctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatata
catatgcatcaccatcaccatcacctcgagggatccgacctcgagatctgcagctggtaccatggaattcgaagcttgatccggctgctaacaaag
cccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctga
aaggaggaactatatccggatctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgc
gccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgcttt
cttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct
cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctt
taatagtggactcttgttccaaactggaacaacactcaaccctatcgcggtctattcttttgatttataagggattttgccgatttcggcctattg
gttaaaaaatgagctgatttaacaaatatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgc
ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg
aagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaa
gtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaa
cgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccccatacac
tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc
atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaact
cgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagagtgacaccacgatgcctgtagcaatgccaacaacgttgcgcaaa
ctattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcc
cttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagcgctcc
cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg
taactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat
ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg
cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaact
ggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc
gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag
cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc
gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc
tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgcc
agcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt
accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaa
ccgcctctccccgcgcgttggccgattcattaatgcag 
Primers para BoHV-5 (Siedler et al., in prep) 
Pretendemos utilizar o plasmídeo pAE 
Inserir o inserto relativo ao fragmento da gE de BoHV-5 nos sítios de 
BamHI: G^GATC_C 
KpnI: G_GTAC^C 
gatctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatata
catatgcatcaccatcaccatcacctcgagggatccgacctcgagatctgcagctggtaccatggaattcgaagcttgatccggctgctaacaaag
cccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctga
aaggaggaactatatccggatctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgc
gccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgcttt 
gatctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatata
catatgcatcaccatcaccatcacctcgaggGATCCGCCCCGGCCGGCGCGGTCTTCACCGCGCGCGTTGGTGCGCCCGTCTTCCTCCCTGGGCCT
GACCCGCGCCCCGAGACGCGCGCCGTTCGCGGCTGGAGCGTCCTCGCGAGCGACTGCCCACCGCCCGAGCCGACGCCCGTCTGCCTCGACGACCGC
GAGTGCTTCGCCGACGTGGCCCTGGACGCGGCCTGCCTGCGGACCGCTCGCATGGCCCCGCTGGCCATCGCAGAGCTCACCGAGCGGCCCGATCCG
GCGGGCGACAGGGAGTTCGTCGTCCCTGACCCGCGCGTTTCCGCGCGGCTGGGCCGCAACGCGACCGGGGTGCAGATCGCGGACGTGACCGAGGAG
GACGGCGGCGTGTACTTCCTGTACGACCGGGCCGCCGGCGACGCCGGCGACGAGGAGACGCAGTCGACCCTGACGCTGCGGGTCGAGCCGGCCGAC
GCTTGGGACCCCGCCGGGCAGGGCGAGGGCGGGGAAGGGGAAGGGGGGAAGGGGGGGCGAGGGGCGGCCAAGCCCACCCCCACCCCCACCCCCGCC
CCCAGCCCGCCCCGCCCCACGCCCGCGCGCCCCGCGCCCCCCCCGCGGCGGCGGCACGGCGCGCGCTTCCGCGTGCAGCCGTACCGCTCCCACGTG
TACACCCCGGGCGACTCCTTCACGCTCTCGGTGCGGCTGCAGTCCGgtaccatggaattcgaagcttgatccggctgctaacaaagcccgaaagga
agctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaac
tatatccggatctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagc
ggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgcttt… 
MHHHHHHLEGSAPAGAVFTARVGAPVFLPGPDPRPETRAVRGWSVLASDCPPPEPTPVCLDDRECFA
DVALDAACLRTARMAPLAIAELTERPDPAGDREFVVPDPRVSARLGRNATGVQIADVTEEDGGVYFLYD
RAAGDAGDEETQSTLTLRVEPADAWDPAGQGEGGEGEGGKGGRGAAKPTPTPTPAPSPPRPTPARPA
PPPRRRHGARFRVQPYRSHVYTPGDSFTLSVRLQSGTMEFEA. 
6xHis gE 
Clonar sequencias de DNA homologas 
• Enguias doentes 
Doente 
Saudável 
Clonar sequencias de DNA homologas• Identificação Herpesvírus na microscópica 
eletrônica 
Clonar sequencias de DNA homologas 
• Regiões conservadas em polimerases de α-β-δ Herpesvírus 
DFAS GDTD 
1235 aa HHV1 
HHV3 1194 aa 
HHV5 1242 aa 
HHV6 
HHV4 
HVS 
1012 aa 
1015 aa 
1009 aa 
Clonar sequencias de DNA homologas 
• Nucleotídeos degenerados 
R = A ou G 
Y = C ou T 
M = A ou C 
K = G ou T 
S = C ou G 
W = A ou T 
 
H = A ou C ou T 
B = C ou G ou T 
V = A ou C ou G 
D = A ou G ou T 
N = A ou C ou G ou T 
Clonar sequencias de DNA homologas 
• Primers degenerados para amplificar o gene da 
DNA polimerase de Enguias 
DFAS GDTD 
1235 aa HHV1 
D F S 
D T D G 
5’GTVBTNGAYTTYSMHAGYYTVTAYCC 3` 
 3’CCNCTRTGBCTRWSVBANAA 5` 
CCV 985 aa 
DFTS GDTD Catfish virus 
Ictalurivirus 
Clonar sequencias de DNA homologas 
• Nova familia: Alloherpesviridae, gênero 
Ictalurivirus 
Rijsewijk, et al., 2005 - J Virol Methods, Vol. 124, No. 1-2, pp. 87-94 
Anguillid herpesvirus 
Clonar sequencias de DNA não conhecidas 
• Por exemplo, a amplificação por círculo rolante 
utilizando a polimerase Phi29 que amplifica DNA 
circulares com o auxilio de primers randômicos 
Sequenciamento de DNA 
• Sequenciamento com o método de Sanger 
DNA plasmidial 
 
 
Produto de PCR 
 
4 tipos ddDNA 
Sequenciamento de DNA 
• Eletroferograma 
Uso de primers como sonda 
Staph. aureus 
Streptococcus 
Mycoplasma 
bovis 
Corynebacterium b. 
Bos taurus 
biotin 
biotin Primer Forward Streptococcus 
Primer Reverse Streptococcus 
Uso de primers como sonda 
Lee, et al. Journal of Veterinary Diagnostic Investigation, v.20, p.463-471, 2008. 
Primers famosos 
• M13 forward e reverse 
M13/pUC sequencing primer (-20), 17-mer 
5'-d(GTAAAACGACGGCCAGT)-3' 
DNA plasmidial 
M13/pUC reverse sequencing primer (-26), 17-mer 
5'-d(CAGGAAACAGCTATGAC)-3' 
M13/pUC sequencing primer (-40), 17-mer 
5'-d(GTTTTCCCAGTCACGAC)-3' 
Outros primers famosos 
• T7 (17-mer) (Stratagene) 5’ AATACGACTCACTATAG 3’ 
T7 (22-mer) (Sratagene) 5’ GTAATACGACTCACTATAGGGC 3’ 
• T3 (17-mer) (Stratagene) 5’ ATTAACCCTCACTAAAG 3’ 
• T3 (20-mer) (Stratagene) 5’ AATTAACCCTCACTAAAGGG 3’ 
• SP6 (Gibco-BRL) 5’ ATTTAGGTGACACTATAG 3’ 
• M13 forward (Stratagene) 5’ GTAAAACGACGGCCAG 3’ 
• M13 forward (Gibco-BRL) 5’ CCCAGTCACGACGTTGTAAAACG 3’ 
• M13 reverse (Stratagene) 5’ GGAAACAGCTATGACCATG 3’ 
• M13 reverse (Gibco-BRL) 5’ AGCGGATAACAATTTCACACAGG 3’ 
• M13 (-20) (Stratagene) 5’ GTAAAACGACGGCCAGT 3’ 
Primers na PCR 
• Reagentes 
– DNA molde 
– Taq polimerase 
– Tampão da enzima 
– MgCl2 
 
– Primers 
– DNTPs 
– H2O 
– Outros: DMSO, glicerol 
• PCR: síntese de DNA in vitro 
 
PCR - Desnaturação 
• Temperatura 
 
Fonte: wps.prenhall.com 
PCR - Anelamento 
PCR - Extensão 
Primers na PCR 
• Ciclos consistindo de 3 etapas: 
 Desnaturação (1-2min a 94ºC) 
 Anelamento (1-2min a 50-55ºC*) 
 Extensão (1-2min** a 72ºC) 
• *Temperatura determinada pela Tm dos 
primers 
• **Tempo determinado pelo tamanho do 
produto 
Primers na PCR 
• *O que é Tm? “Melting temperature” 
50% fita dupla 
50% desnaturada 
 
Primers na PCR 
• A característica da sequência alvo também 
vai determinar a Tm 
• P. ex.: Herpesvírus bovino 1 ou 5 apresentam 
um conteúdo de GC em torno de 72% 
• Herpesvírus ovino tipo 2: 52% de GC 
• SV 40 (poliomavírus): 40% GC 
Desenho de primers 
• Desenhados para serem complementares 
a sequência alvo 
 Anelar numa região única do genoma 
• Características desejáveis: 
 Tamanho: entre 15-30pb 
 Conteúdo: 50% de GC 
 Tm similar 
 
Desenho de primers 
• Características desejáveis: 
 Extremidade 3’ deve conter 2-3 bases GC: 
aumentar a fidelidade de anelamento 
 Não deve possuir um conjunto de bases que 
permita o “self-annealing” ou formação de 
“loops” 
 Evitar a formação de dímeros de primers 
 
• Dímeros de primers 
Desenho de primers 
Desenho de primers 
• Outras observações: 
 Baixa Tm: falha no anelamento e 
produtos de tamanho diferente ao 
esperado (bandas inespecíficas) 
 Alta Tm: pode falhar no anelamento e/ou 
estender continuamente (Taq polimerase 
temperatura de 72oC) 
 
Desenho de primers 
Site para download: 
http://www.genelink.com/tools/OEreg.asp 
OLIGO EXPLORER 
Desenho de primers 
OLIGO ANALYZER 
• Sequência alvo 
Desenho de primers 
• Sequência alvo 
Desenho de primers 
TATGGAGAAGGAAGAGCCCGATACGCTCGCACCACGAGCTTCACGTGACGCTCCGGGG
ACGCCAAAAGTGCCCGCGATGCCCGGTGTGACCCCGGAGCCATCAGGAAACGCCTCG
GAGCCCGCCGACCCGGCGGAGCTCCGGGCGGACTTGCGGGGTCTAAAAGGCTCCAG
CGACGATCCTAATTTCTACGTGTGCCCCCCTCCCACCGGTGCGACCGTGGTGCGGCTC
GAGGAGCCGCGCCCGTGCCCGGAGTTGCCCAAGGGGCTCAACTTCACCGAGGGCATC
GCCGTAACTTTCAAGGAGAACCTCGCGCCGTACAAGTTTAAGGCCACTATGTACTACAAG
GCCGTGACTGTCGCTAGCGTGTGGTCCGGGTACTCGTATAACCAGTTTATGAATATCTTT
GAGGACCGTGCCCCTATACCGTTCGAGGAAATCGTCGACCGGATACACGGCCGGGGTA
TGTGTTTGTCCACGGCAAAGTACGTCAGGAACAACCTTGAGACCACCGCATTCCATAAT
GATGCCGACGAACATGAGATGAAGCTGGTGCCCGCCGAGTCCGCCCCTGGGTTGCATC
GCGGATGGCACACCACGCGGCTAAAAA 
• Sequência alvo: Bovine herpesvirus type 2 
glycoprotein B gene 
Desenho de primers 
Desenho de primers 
Desenho de primers 
PRIMER FORWARD 
(Upper primer) 
Desenho de primers 
PRIMER REVERSE 
(Lower primer) 
Desenho de primers 
Características do 
Primer forward 
Desenho de primers 
Características do 
Primer reverse 
Desenho de primers 
Formação de dímeros 
Primers de um artigo 
A1: 5’-GAGAGACTTGAAGATGTCTTTGCTG-3’ (Tm:64oC, GC%: 44,0%) 
A2: 5’-GCTCTGTCCATGTTATTTGGATC-3‘ (Tm:61,1oC, GC%: 43,5%) 
Formação de dímeros 
Blast 
Blast 
Blast 
Blast 
Tamanho do produto 
1 2 
4 
3 
Bovine herpesvirus type 2 glycoprotein B gene 
BoHV-2F: TATGGAGAAGGAAGAGCCCG 
BoHV-2R: TTTTTAGCCGCGTGGTGTGC 
TATGGAGAAGGAAGAGCCCGATACGCTCGCACCACGAGCTTCACGTGACGCTCCGGG
GACGCCAAAAGTGCCCGCGATGCCCGGTGTGACCCCGGAGCCATCAGGAAACGCCTC
GGAGCCCGCCGACCCGGCGGAGCTCCGGGCGGACTTGCGGGGTCTAAAAGGCTCCA
GCGACGATCCTAATTTCTACGTGTGCCCCCCTCCCACCGGTGCGACCGTGGTGCGGCT
CGAGGAGCCGCGCCCGTGCCCGGAGTTGCCCAAGGGGCTCAACTTCACCGAGGGCAT
CGCCGTAACTTTCAAGGAGAACCTCGCGCCGTACAAGTTTAAGGCCACTATGTACTACAA
GGCCGTGACTGTCGCTAGCGTGTGGTCCGGGTACTCGTATAACCAGTTTATGAATATCTT
TGAGGACCGTGCCCCTATACCGTTCGAGGAAATCGTCGACCGGATACACGGCCGGGGT
ATGTGTTTGTCCACGGCAAAGTACGTCAGGAACAACCTTGAGACCACCGCATTCCATAAT
GATGCCGACGAACATGAGATGAAGCTGGTGCCCGCCGAGTCCGCCCCTGGGTTGCATC
GCGGATGGCACACCACGCGGCTAAAAA 
Produto da PCR: 
 608 pb 
 
• Sequência alvo vai dar origem ao produto de 
PCR 
 
Desenho de primers 
Considerações finais 
• O desenho dos primers com as 
carcterísticas ideais reflete em menos tempo 
de padronização da PCR 
 Cada nucleotídeo diferente dá uma perda de 
sensibilidade de aproximadamente 10 vezes 
• Sempre vai depender da finalidade 
 Diagnóstico (sensibilidade) 
 
Considerações finais 
 Amplificação de 1 gene específico para 
fins de expressão / clonagem 
• Região específica de um gene ou o ínicio e 
o fim de um gene (características ideais 
ficam comprometidas) 
• Conhecer a sequencia alvo e o tamanho do 
produto é fundamental na amplificação de 
genes 
 
Referências 
• Donofrio, J.C., Coonrod, J.D., Davidson, J.N., Betts, R.F. Detection of Influenza A and 
B in Respiratory Secretions with the Polymerase Chain Reaction. Genome Res, v.1, 
p.263-268, 1992. 
• Kamstrup, S., Barfoed, A.M.,Frimann, T.H., Ladekjær-Mikkelsen, A.S., Bøtner, A. 
Immunisation against PCV2 structural protein by DNA vaccination of mice. Vaccine, v. 
22, p.1358-1361, 2004. 
• Lee, K.H., Lee, J.W., Wang, S.W., Liu, L.Y., Lee, M.F., Chuang, S.T., Shy, Y.M., 
Chang, C.L., Wu, M.C., Chi, C.H. Development of a novel biochip for rapid multiplex 
detection of seven mastitis-causing pathogens in bovine milk samples. Journal of 
Veterinary Diagnostic Investigation, v.20, p.463-471, 2008. 
• Rijsewijk, F., Pritz-Verschuren, S., Kerkhoff, S., Botter, A., Willemsen, M., Nieuwstadt, 
T., Haenen, O. Development of a polymerase chain reaction for the detection of 
Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene 
Journal of Virological Methods, v.124, p. 87–94, 2005. 
Vamos praticar 
1o Acessar o http://www.ncbi.nlm.nih.gov/ 
 Alterar para Nucleotide 
2º Buscar a sequencia alvo 
 Sugestão: Chicken Parvovirus complete genome 
3º Clicar em Chicken parvovirus ABU-P1,... 
 Alterar para formato Fasta 
4º Copiar a sequencia 
5º Abrir o Oligo Explorer 
 
 
Continuando… 
5º Abrir o Oligo Explorer 
 Clicar em New 
 Colar a sequencia e clicar em Ok 
 Desenhar primers para amplificar todo o genoma: 
PF e PR 
6º Abrir o Oligo Analyzer: checar os primers 
7º Fazer o Blast: http://blast.ncbi.nlm.nih.gov/ PF e PR 
8º Determinar o tamanho do produto 
 
 
 
Grato pela atenção! 
Contato: 
camposvet@gmail.com