biologia molecular , revisão do conteúdo

Biologia Molecular, revisão do conteúdo

Prof Francisco Prosdocimi

ÁCIDOS

NUCLEICOS

DNA, RNA

Armazenamento da informação genética

Polímeros de nucleotídeos

DNA E RNA

Polímeros de nucleotídeos

Esqueleto de ribose-fosfato ligado às bases nitrogenadas

REPLICAÇÃO

DO DNA

O DNA é composto por uma dupla-hélice

Replicação semi-conservativa: as bases presentes em uma das fitas contém toda a informação necessária para a síntese da nova fita

A complementaridade das bases A = T, G = C

As duas fitas do DNA são antiparalelas

EVOLUÇÃO POR MUTAÇÕES

A modificação das moléculas de DNA ao longo do tempo (mutação) é um dos principais fatores evolutivos

DOGMA CENTRAL E TRADUÇÃO

PROTEÍNAS

Moléculas mais importantes?

Polímeros de aminoácidos

Apenas 20 diferentes aminoácidos estão presentes nas moléculas biológicas

Carboxil

Amino

AMINOÁCIDOS

LIGANDO AMINOÁCIDOS

Onde acontece? Quem atua como

catalisador?

ESTRUTURA DAS PROTEÍNAS

Enovelamento de proteínas

HIERARQUIA ESTRUTURAL

ALFABETO QUÍMICO

Todos os organismos vivos são constituídos a partir das mesmas unidades monoméricas

A estrutura das macromoléculas é o que determina a sua função biológica

Cada espécie apresenta um conjunto distinto de macromoléculas

O Sequenciamento de moléculas de DNAProf. Dr. Francisco Prosdocimi

>gi|33869444|gb|BC008730.2| Homo sapiens hexokinase 1, mRNA (cDNA clone MGC:1724 IMAGE:3163058), complete cds GGCTGCGGAGGACCGACCGTCCCCACGCCTGCCGCCCCGCGACCCCGACCGCCAGCATGATCGCCGCGCA GCTCCTGGCCTATTACTTCACGGAGCTGAAGGATGACCAGGTCAAAAAGATTGACAAGTATCTGTATGCC ATGCGGCTCTCCGATGAAACTCTCATAGATATCATGACTCGCTTCAGGAAGGAGATGAAGAATGGCCTCT CCCGGGATTTTAATCCAACAGCCACAGTCAAGATGTTGCCAACATTCGTAAGGTCCATTCCTGATGGCTC TGAAAAGGGAGATTTCATTGCCCTGGATCTTGGTGGGTCTTCCTTTCGAATTCTGCGGGTGCAAGTGAAT CATGAGAAAAACCAGAATGTTCACATGGAGTCCGAGGTTTATGACACCCCAGAGAACATCGTGCACGGCA GTGGAAGCCAGCTTTTTGATCATGTTGCTGAGTGCCTGGGAGATTTCATGGAGAAAAGGAAGATCAAGGA CAAGAAGTTACCTGTGGGATTCACGTTTTCTTTTCCTTGCCAACAATCCAAAATAGATGAGGCCATCCTG ATCACCTGGACAAAGCGATTTAAAGCGAGCGGAGTGGAAGGAGCAGATGTGGTCAAACTGCTTAACAAAG(...)TGACAGGCCTTCTGGGCCTCCAAAGCCCATCCTTGGGGTTCCCCCTCCCTGTGTGAAATGTATTATCACCAGCAGACACTGCCGGGCCTCCCTCCCGGGGGCACTGCCTGAAGGCGAGTGTGGGCATAGCATTAGCTGCT TCCTCCCCTCCTGGCACCCACTGTGGCCTGGCATCGCATCGTGGTGTGTCAATGCCACAAAATCGTGTGT CCGTGGAACCAGTCCTAGCCGCGTGTGACAGTCTTGCATTCTGTTTGTCTCGTGGGGGGAGGTGGACAGT CCTGCGGAAATGTGTCTTGTCTCCATTTGGATAAAAGGAACCAACCAACAAACAATGCCATCACTGGAAT TTCCCACCGCTTTGTGAGCCGTGTCGTATGACCTAGTAAACTTTGTACCAATTCAAAAAAAAAAAAAAAAAA

Bioquímica + Biomol

• Enzimas são proteínas, portanto:

1. São formadas por sequências de aminoácidos

2. Derivam de informações dispostas por genes no DNA, que deve ser transcrito e, posteriormente, traduzido

3. Podemos saber a sequência delas, tanto de aminoácidos quanto de nucleotídeos

>gi|188497753|ref|NM_000188.2| Homo sapiens hexokinase 1 (HK1), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA GAGGAGGAGCCGCCGAGCAGCCGCCGGAGGACCACGGCTCGCCAGGGCTGCGGAGGACCGACCGTCCCCA CGCCTGCCGCCCCGCGACCCCGACCGCCAGCATGATCGCCGCGCAGCTCCTGGCCTATTACTTCACGGAG CTGAAGGATGACCAGGTCAAAAAGATTGACAAGTATCTCTATGCCATGCGGCTCTCCGATGAAACTCTCA TAGATATCATGACTCGCTTCAGGAAGGAGATGAAGAATGGCCTCTCCCGGGATTTTAATCCAACAGCCAC AGTCAAGATGTTGCCAACATTCGTAAGGTCCATTCCTGATGGCTCTGAAAAGGGAGATTTCATTGCCCTG GATCTTGGTGGGTCTTCCTTTCGAATTCTGCGGGTGCAAGTGAATCATGAGAAAAACCAGAATGTTCACA TGGAGTCCGAGGTTTATGACACCCCAGAGAACATCGTGCACGGCAGTGGAAGCCAGCTTTTTGATCATGT TGCTGAGTGCCTGGGAGATTTCATGGAGAAAAGGAAGATCAAGGACAAGAAGTTACCTGTGGGATTCACG TTTTCTTTTCCTTGCCAACAATCCAAAATAGATGAGGCCATCCTGATCACCTGGACAAAGCGATTTAAAG CGAGCGGAGTGGAAGGAGCAGATGTGGTCAAACTGCTTAACAAAGCCATCAAAAAGCGAGGGGACTATGA TGCCAACATCGTAGCTGTGGTGAATGACACAGTGGGCACCATGATGACCTGTGGCTATGACGACCAGCAC TGTGAAGTCGGCCTGATCATCGGCACTGGCACCAATGCTTGCTACATGGAGGAACTGAGGCACATTGATC TGGTGGAAGGAGACGAGGGGAGGATGTGTATCAATACAGAATGGGGAGCCTTTGGAGACGATGGATCATT AGAAGACATCCGGACAGAGTTTGACAGGGAGATAGACCGGGGATCCCTCAACCCTGGAAAACAGCTGTTT GAGAAGATGGTCAGTGGCATGTACTTGGGAGAGCTGGTTCGACTGATCCTAGTCAAGATGGCCAAGGAGG GCCTCTTATTTGAAGGGCGGATCACCCCGGAGCTGCTCACCCGAGGGAAGTTTAACACCAGTGATGTGTC AGCCATCGAAAAGAATAAGGAAGGCCTCCACAATGCCAAAGAAATCCTGACCCGCCTGGGAGTGGAGCCG TCCGATGATGACTGTGTCTCAGTCCAGCACGTTTGCACCATTGTCTCATTTCGCTCAGCCAACTTGGTGG CTGCCACACTGGGCGCCATCTTGAACCGCCTGCGTGATAACAAGGGCACACCCAGGCTGCGGACCACGGT TGGTGTCGACGGATCTCTTTACAAGACGCACCCACAGTATTCCCGGCGTTTCCACAAGACTCTAAGGCGC TTGGTGCCAGACTCCGATGTGCGCTTCCTCCTCTCGGAGAGTGGCAGCGGCAAGGGGGCTGCCATGGTGA CGGCGGTGGCCTACCGCTTGGCCGAGCAGCACCGGCAGATAGAGGAGACCCTGGCTCATTTCCACCTCAC CAAGGACATGCTGCTGGAGGTGAAGAAGAGGATGCGGGCCGAGATGGAGCTGGGGCTGAGGAAGCAGACG CACAACAATGCCGTGGTTAAGATGCTGCCCTCCTTCGTCCGGAGAACTCCCGACGGGACCGAGAATGGTG ACTTCTTGGCCCTGGATCTTGGAGGAACCAATTTCCGTGTGCTGCTGGTGAAAATCCGTAGTGGGAAAAA GAGAACGGTGGAAATGCACAACAAGATCTACGCCATTCCTATTGAAATCATGCAGGGCACTGGGGAAGAG CTGTTTGATCACATTGTCTCCTGCATCTCTGACTTCTTGGACTACATGGGGATCAAAGGCCCCAGGATGC CTCTGGGCTTCACGTTCTCATTTCCCTGCCAGCAGACGAGTCTGGACGCGGGAATCTTGATCACGTGGAC AAAGGGTTTTAAGGCAACAGACTGCGTGGGCCACGATGTAGTCACCTTACTAAGGGATGCGATAAAAAGG AGAGAGGAATTTGACCTGGACGTGGTGGCTGTGGTCAACGACACAGTGGGCACCATGATGACCTGTGCTT ATGAGGAGCCCACCTGTGAGGTTGGACTCATTGTTGGGACCGGCAGCAATGCCTGCTACATGGAGGAGAT GAAGAACGTGGAGATGGTGGAGGGGGACCAGGGGCAGATGTGCATCAACATGGAGTGGGGGGCCTTTGGG GACAACGGGTGTCTGGATGATATCAGGACACACTACGACAGACTGGTGGACGAATATTCCCTAAATGCTG GGAAACAAAGGTATGAGAAGATGATCAGTGGTATGTACCTGGGTGAAATCGTCCGCAACATCTTAATCGA CTTCACCAAGAAGGGATTCCTCTTCCGAGGGCAGATCTCTGAGACGCTGAAGACCCGGGGCATCTTTGAG ACCAAGTTTCTCTCTCAGATCGAGAGTGACCGATTAGCACTGCTCCAGGTCCGGGCTATCCTCCAGCAGC TAGGTCTGAATAGCACCTGCGATGACAGTATCCTCGTCAAGACAGTGTGCGGGGTGGTGTCCAGGAGGGC CGCACAGCTGTGTGGCGCAGGCATGGCTGCGGTTGTGGATAAGATCCGCGAGAACAGAGGACTGGACCGT CTGAATGTGACTGTGGGAGTGGACGGGACACTCTACAAGCTTCATCCACACTTCTCCAGAATCATGCACC AGACGGTGAAGGAACTGTCACCAAAATGTAACGTGTCCTTCCTCCTGTCTGAGGATGGCAGCGGCAAGGG GGCCGCCCTCATCACGGCCGTGGGCGTGCGGTTACGCACAGAGGCAAGCAGCTAAGAGTCCGGGATCCCC AGCCTACTGCCTCTCCAGCACTTCTCTCTTCAAGCGGCGACCCCCTACCCTCCCAGCGAGTTGCGCTGGG AGACGCTGGCGCCAGGGCCTGCCGGCGCGGGGAGGAAAGCAAAATCCAACTAATGGTATATATTGTAGGG TACAGAATAGAGCGTGTGCTGTTGATAATATCTCTCACCCGGATCCCTCCTCACTTGCCCTGCCACTTTG CATGGTTTGATTTTGACCTGGTCCCCCACGTGTGAAGTGTAGTGGCATCCATTTCTAATGTATGCATTCA TCCAACAGAGTTATTTATTGGCTGGAGATGGAAAATCACACCACCTGACAGGCCTTCTGGGCCTCCAAAG CCCATCCTTGGGGTTCCCCCTCCCTGTGTGAAATGTATTATCACCAGCAGACACTGCCGGGCCTCCCTCC CGGGGGCACTGCCTGAAGGCGAGTGTGGGCATAGCATTAGCTGCTTCCTCCCCTCCTGGCACCCACTGTG GCCTGGCATCGCATCGTGGTGTGTCAATGCCACAAAATCGTGTGTCCGTGGAACCAGTCCTAGCCGCGTG TGACAGTCTTGCATTCTGTTTGTCTCGTGGGGGGAGGTGGACAGTCCTGCGGAAATGTGTCTTGTCTCCA TTTGGATAAAAGGAACCAACCAACAAACAATGCCATCACTGGAATTTCCCACCGCTTTGTGAGCCGTGTC GTATGACCTAGTAAACTTTGTACCAATTCAAAAAAAAAAAAAAAAAA

>gi|188497754|ref|NP_000179.2| hexokinase 1 isoform HKI [Homo sapiens] MIAAQLLAYYFTELKDDQVKKIDKYLYAMRLSDETLIDIMTRFRKEMKNGLSRDFNPTATVKMLPTFVRS IPDGSEKGDFIALDLGGSSFRILRVQVNHEKNQNVHMESEVYDTPENIVHGSGSQLFDHVAECLGDFMEK RKIKDKKLPVGFTFSFPCQQSKIDEAILITWTKRFKASGVEGADVVKLLNKAIKKRGDYDANIVAVVNDT VGTMMTCGYDDQHCEVGLIIGTGTNACYMEELRHIDLVEGDEGRMCINTEWGAFGDDGSLEDIRTEFDRE IDRGSLNPGKQLFEKMVSGMYLGELVRLILVKMAKEGLLFEGRITPELLTRGKFNTSDVSAIEKNKEGLH NAKEILTRLGVEPSDDDCVSVQHVCTIVSFRSANLVAATLGAILNRLRDNKGTPRLRTTVGVDGSLYKTH PQYSRRFHKTLRRLVPDSDVRFLLSESGSGKGAAMVTAVAYRLAEQHRQIEETLAHFHLTKDMLLEVKKR MRAEMELGLRKQTHNNAVVKMLPSFVRRTPDGTENGDFLALDLGGTNFRVLLVKIRSGKKRTVEMHNKIY AIPIEIMQGTGEELFDHIVSCISDFLDYMGIKGPRMPLGFTFSFPCQQTSLDAGILITWTKGFKATDCVG HDVVTLLRDAIKRREEFDLDVVAVVNDTVGTMMTCAYEEPTCEVGLIVGTGSNACYMEEMKNVEMVEGDQ GQMCINMEWGAFGDNGCLDDIRTHYDRLVDEYSLNAGKQRYEKMISGMYLGEIVRNILIDFTKKGFLFRG QISETLKTRGIFETKFLSQIESDRLALLQVRAILQQLGLNSTCDDSILVKTVCGVVSRRAAQLCGAGMAA VVDKIRENRGLDRLNVTVGVDGTLYKLHPHFSRIMHQTVKELSPKCNVSFLLSEDGSGKGAALITAVGVR LRTEASS

917 aminoácidos

3617 bp3,6 kb

917 x 3 = 2751

3617-2751 = 866

O método de Sanger, 1975

Polimerização do DNA a ser sequenciado (molde)na presença de:

DNA polimeraseprimer

tampãodNTPs (desóxinucleotídeo)ddNTPs (didesóxinucleotídeo)

O que faria um nucleotídeo que, ao invés da extremidade 3’OH, tem uma extremidade 3’H?

Como acontece a síntese de moléculas de DNA?

http://www.youtube.com/watch?v=Mz-4LSfecM4&feature=related (dideóxi)

http://www.youtube.com/watch?v=Mz-4LSfecM4&feature=related

http://www.youtube.com/watch?v=Mz-4LSfecM4&feature=related

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||

5’3’

ATGCTTCATGCTTC5’ 3’

AA

AAAA

AA

AA

AA

AAAA

AA

AA

TT TTTT TT

TT TTTT

TT

TT

TTTT

GG

GG

GGGG

GG

GG

GG

GG

GG

CC

CC

CC

CC

CC

CC

CC

CC

CCCC

CC

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||||

5’3’

ATGCTTCATGCTTC5’ 3’

AA

AA

AA

AA

AAAA

AAAA

AA

AA

TTTTTT TT

TT TTTT

TT

TT

TTTT

GG

GG

GGGGGG

GG

GG

GGGG

CC

CC

CC

CCCC

CC

CC

CC

CC

CC

CC

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA||||||||||

3’

ATGCTTCATGCTTCTGTG5’ 3’

AA

AAAA

AA

AA AA

AAAA

AA

AA

TT TTTT

TT

TT TTTT

TT

TT

TTTT

GG

GG

GGGG

GG

GG

GG

GG

GG

CC

CCCC

CC

CC

CC

CC

CC

CCCC

CC

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||||||||||||

5’3’

ATGCTTCATGCTTCTGGCAGATCTTGGCAGATCT5’ 3’

AA

AAAA

AA

AAAA

AA

AA

AA AA

TT TTTT

TT

TTTTTT

TT

TT

TTTT GGGG

GGGG GG

GG

GG

GG GG

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

CC

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||||||||||

5’3’

ATGCTTCATGCTTCTGGCAGATGGCAGATT5’ 3’

AA

AA

AAAA

AA

AA

AAAA

AA

AA

TTTTTT

TTTT

TTTTTT

TTTT

TT

GG

GGGG

GG

GGGG

GG

GG GG

CCCCCC

CC CCCC

CC

CCCC

CC

CC


5’3’


AA

AA

AA AAAA

AA

AAAA

AA

AATT

TTTT

TT

TTTTTTTT

TT TT

TTGG

GGGG

GG

GGGG

GG

GG GG

CC

CCCC

CCCC

CC

CC

CC

CCCC

CC


5’3’


AA

AA

AAAA

AA

AA

AAAA

AA

AA TT

TTTT

TTTTTT

TTTT

TTTT

TTGG

GGGG

GG

GGGG

GG

GGGG

CC

CCCC

CCCC

CC

CC

CCCC

CC

CC

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATGGCAGATCTGAACAGTGTTACTGATT5’ 3’

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTGGCAGATCTGAACAGTGTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTGGCAGATCTGAACAGTGTTACTGATATTGCTTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATGGCAGATCTGAACAGTGTTACTGATATT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGGCAGATCTGAACAGTGTTACTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGGCAGATCTGAACAGTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTGGCAGATCTGAACAGTGTTACTGATATTGCTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGGCAGATCTGAACAGTGTTACTGATATTT

ATGCTTCATGCTTCTT

ATGCTTCATGCTTCTGGCAGATCTGGCAGATCTT

TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA||||||||||||||||||||||||||||||||||||||||

5’3’

ATGCTTCATGCTTCTGGCAGATGGCAGATT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTTGGCAGATCTGAACAGTGTTACTGATATTGCTT

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTGGCAGATCTGAACAGTGTTT

População de moléculas

Incorporação aleatória do didesóxi

Quantidade precisa entre didesóxi e desóxi


ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATGGCAGATCTGAACAGTGTTACTGATT






ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTGGCAGATCTGAACAGTGTTACTGATATTGCTTATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGGCAGATCTGAACAGTGTTACTGATATTT

ATGCTTCATGCTTCTT




G A T C

• moldemolde• polimerasepolimerase• dNTPsdNTPs

•ddGTPsddGTPs •ddATPsddATPs •ddTTPsddTTPs •ddCTPsddCTPs










ATGCTTCATGCTTCTT




ATGCTTCATGCTTCTTGGATGCTTCATGCTTCTGTGGG

ATGCTTCATGCTTCTGGCATGGCAGG

ATGCTTCATGCTTCTGGCAGATCTTGGCAGATCTGG

ATGCTTCATGCTTCTGGCAGATCTGAACATGGCAGATCTGAACAGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTTGGCAGATCTGAACAGTGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTTGGCAGATCTGAACAGTGTTACTGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTTGGCAGATCTGAACAGTGTTACTGATATTGG

ATGCTTCATGCTTCTGGTGGCC

ATGCTTCATGCTTCTGGCAGATTGGCAGATCC

ATGCTTCATGCTTCTGGCAGATCTGAATGGCAGATCTGAACC

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTATGGCAGATCTGAACAGTGTTACC

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGTGGCAGATCTGAACAGTGTTACTGATATTGCC

ATGCTTCATGCTTCTGGCTGGCAA

ATGCTTCATGCTTCTGGCAGTGGCAGAA

ATGCTTCATGCTTCTGGCAGATCTGTGGCAGATCTGAAATGCTTCATGCTTCTGGCAGATCTGATGGCAGATCTGAAA

ATGCTTCATGCTTCTGGCAGATCTGAACTGGCAGATCTGAACAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTTGGCAGATCTGAACAGTGTTAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGTGGCAGATCTGAACAGTGTTACTGAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATTGGCAGATCTGAACAGTGTTACTGATAA

G A T C










ATGCTTCATGCTTCTT




ATGCTTCATGCTTCTTGGATGCTTCATGCTTCTGTGGG

ATGCTTCATGCTTCTGGCATGGCAGG

ATGCTTCATGCTTCTGGCAGATCTTGGCAGATCTGG

ATGCTTCATGCTTCTGGCAGATCTGAACATGGCAGATCTGAACAGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTTGGCAGATCTGAACAGTGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTTGGCAGATCTGAACAGTGTTACTGG

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTTGGCAGATCTGAACAGTGTTACTGATATTGG

ATGCTTCATGCTTCTGGTGGCC

ATGCTTCATGCTTCTGGCAGATTGGCAGATCC

ATGCTTCATGCTTCTGGCAGATCTGAATGGCAGATCTGAACC

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTATGGCAGATCTGAACAGTGTTACC

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGTGGCAGATCTGAACAGTGTTACTGATATTGCC

ATGCTTCATGCTTCTGGCTGGCAA

ATGCTTCATGCTTCTGGCAGTGGCAGAA

ATGCTTCATGCTTCTGGCAGATCTGTGGCAGATCTGAAATGCTTCATGCTTCTGGCAGATCTGATGGCAGATCTGAAA

ATGCTTCATGCTTCTGGCAGATCTGAACTGGCAGATCTGAACAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTTGGCAGATCTGAACAGTGTTAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGTGGCAGATCTGAACAGTGTTACTGAA

ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATTGGCAGATCTGAACAGTGTTACTGATAA

As máquinas necessárias para o sequenciamento

• Primeira etapa: junta-se os reagentes em poços de placas e coloca-se na máquina de PCR para a reaçãode amplificação interrompida

• Diferenças com relação ao PCR– Utilização de um só primer– Utilização dos ddNTPs

• Uma vez prontas, as sequências de diferentes tamanhos contendo os didesóxi amplificados devem ser enviadas ao sequenciador de DNA mais próximo

O que faz um sequenciador de DNA?

• Segunda etapa: realiza a eletroforese capilar– O sequenciador executa a eletroforese em géis capilares ultra-finos– Um sensor é responsável por emitir um laser e verificar qual o comprimento

de onda emitido pelo didesóxi

A produção de bibliotecas de DNA e cDNA

Prof. Dr. Francisco Prosdocimi

Projetos Genoma e Transcriptoma

O que é um genoma?• Conjunto haplóide de informações

presentes no DNA de determinado organismo

– Genomas bacterianos X Genomas eucarióticos

• Cromossomos são formados por uma única molécula de DNA

– Genoma humano: 22 pares de cromossomos autossomos + X + Y

• O problema da variação – SNPs

• Estudos genômicos e o método científico

– A era da pesquisa científica sem hipótese

Por que haplóide?

Biblioteca de DNA e cDNADNA

Fragmentação

Inserção em vetores

Transformação

mRNA

Síntese de cDNA

Inserção em vetores

Transformação

Biblioteca transcriptômica

• Ou biblioteca de cDNA, DNA complementar

• Purificação dos mRNAs– Oligos dT

• Retrotranscrição

• Clonagem

Análises genômicas e transcriptômicas

• Genoma: muito utilizado para produzir sequências completas do DNA de bactérias e vírus, que apresentam genoma compacto

– Assim é possível saber se o organismo tem as vias bioquímicas completas e como ele deve se alimentar

• Transcriptoma: classicamente utilizado em estudos de células cancerosas, onde a diferença na expressão dos genes deve mostrar porque a célula é tumoral

– Comparação entre a expressão gênica em uma célula normal e o tumor

– Comparações quaisquer entre dois estados celulares

http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome

Expressão gênica

Bioinformática, formatos de arquivo

O formato FASTA• Fast Alignment: programa de alinhamento da

década de 80• Arquivo texto• FASTA e multi-FASTA

>gi|188497754|ref|NP_000179.2| hexokinase 1 isoform HKI [Homo sapiens] MIAAQLLAYYFTELKDDQVKKIDKYLYAMRLSDETLIDIMTRFRKEMKNGLSRDFNPTATVKMLPTFVRS IPDGSEKGDFIALDLGGSSFRILRVQVNHEKNQNVHMESEVYDTPENIVHGSGSQLFDHVAECLGDFMEK RKIKDKKLPVGFTFSFPCQQSKIDEAILITWTKRFKASGVEGADVVKLLNKAIKKRGDYDANIVAVVNDT VGTMMTCGYDDQHCEVGLIIGTGTNACYMEELRHIDLVEGDEGRMCINTEWGAFGDDGSLEDIRTEFDRE IDRGSLNPGKQLFEKMVSGMYLGELVRLILVKMAKEGLLFEGRITPELLTRGKFNTSDVSAIEKNKEGLH NAKEILTRLGVEPSDDDCVSVQHVCTIVSFRSANLVAATLGAILNRLRDNKGTPRLRTTVGVDGSLYKTH PQYSRRFHKTLRRLVPDSDVRFLLSESGSGKGAAMVTAVAYRLAEQHRQIEETLAHFHLTKDMLLEVKKR MRAEMELGLRKQTHNNAVVKMLPSFVRRTPDGTENGDFLALDLGGTNFRVLLVKIRSGKKRTVEMHNKIY AIPIEIMQGTGEELFDHIVSCISDFLDYMGIKGPRMPLGFTFSFPCQQTSLDAGILITWTKGFKATDCVG HDVVTLLRDAIKRREEFDLDVVAVVNDTVGTMMTCAYEEPTCEVGLIVGTGSNACYMEEMKNVEMVEGDQ GQMCINMEWGAFGDNGCLDDIRTHYDRLVDEYSLNAGKQRYEKMISGMYLGEIVRNILIDFTKKGFLFRG QISETLKTRGIFETKFLSQIESDRLALLQVRAILQQLGLNSTCDDSILVKTVCGVVSRRAAQLCGAGMAA VVDKIRENRGLDRLNVTVGVDGTLYKLHPHFSRIMHQTVKELSPKCNVSFLLSEDGSGKGAALITAVGVR LRTEASS

>gi|188497753|ref|NM_000188.2| Homo sapiens hexokinase 1 (HK1), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA GAGGAGGAGCCGCCGAGCAGCCGCCGGAGGACCACGGCTCGCCAGGGCTGCGGAGGACCGACCGTCCCCA CGCCTGCCGCCCCGCGACCCCGACCGCCAGCATGATCGCCGCGCAGCTCCTGGCCTATTACTTCACGGAG CTGAAGGATGACCAGGTCAAAAAGATTGACAAGTATCTCTATGCCATGCGGCTCTCCGATGAAACTCTCA TAGATATCATGACTCGCTTCAGGAAGGAGATGAAGAATGGCCTCTCCCGGGATTTTAATCCAACAGCCAC AGTCAAGATGTTGCCAACATTCGTAAGGTCCATTCCTGATGGCTCTGAAAAGGGAGATTTCATTGCCCTG GATCTTGGTGGGTCTTCCTTTCGAATTCTGCGGGTGCAAGTGAATCATGAGAAAAACCAGAATGTTCACA TGGAGTCCGAGGTTTATGACACCCCAGAGAACATCGTGCACGGCAGTGGAAGCCAGCTTTTTGATCATGT TGCTGAGTGCCTGGGAGATTTCATGGAGAAAAGGAAGATCAAGGACAAGAAGTTACCTGTGGGATTCACG TTTTCTTTTCCTTGCCAACAATCCAAAATAGATGAGGCCATCCTGATCACCTGGACAAAGCGATTTAAAG CGAGCGGAGTGGAAGGAGCAGATGTGGTCAAACTGCTTAACAAAGCCATCAAAAAGCGAGGGGACTATGA TGCCAACATCGTAGCTGTGGTGAATGACACAGTGGGCACCATGATGACCTGTGGCTATGACGACCAGCAC TGTGAAGTCGGCCTGATCATCGGCACTGGCACCAATGCTTGCTACATGGAGGAACTGAGGCACATTGATC TGGTGGAAGGAGACGAGGGGAGGATGTGTATCAATACAGAATGGGGAGCCTTTGGAGACGATGGATCATT

>gi|188497753|ref|NM_000188.2| Homo sapiens hexokinase 1 (HK1), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA

CAAGGACATGCTGCTGGAGGTGAAGAAGAGGATGCGGGCCGAGATGGAGCTGGGGCTGAGGAAGCAGACG CACAACAATGCCGTGGTTAAGATGCTGCCCTCCTTCGTCCGGAGAACTCCCGACGGGACCGAGAATGGTG ACTTCTTGGCCCTGGATCTTGGAGGAACCAATTTCCGTGTGCTGCTGGTGAAAATCCGTAGTGGGAAAAA GAGAACGGTGGAAATGCACAACAAGATCTACGCCATTCCTATTGAAATCATGCAGGGCACTGGGGAAGAG CTGTTTGATCACATTGTCTCCTGCATCTCTGACTTCTTGGACTACATGGGGATCAAAGGCCCCAGGATGC CTCTGGGCTTCACGTTCTCATTTCCCTGCCAGCAGACGAGTCTGGACGCGGGAATCTTGATCACGTGGAC AAAGGGTTTTAAGGCAACAGACTGCGTGGGCCACGATGTAGTCACCTTACTAAGGGATGCGATAAAAAGG AGAGAGGAATTTGACCTGGACGTGGTGGCTGTGGTCAACGACACAGTGGGCACCATGATGACCTGTGCTT

O formato GenBank

• http://www.ncbi.nlm.nih.gov

• Comandos LINUX

http://www.ncbi.nlm.nih.gov/

biologia molecular , revisão do conteúdo

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