Download 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 semiconservativa: 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 DNA
Prof. 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(...)
TGACAGGCCTTCTGGGCCTCCAAAGCCCATCCTTGGGGTTCCCCCTCCCTGTGTGAAATGTATTATCACC
AGCAGACACTGCCGGGCCTCCCTCCCGGGGGCACTGCCTGAAGGCGAGTGTGGGCATAGCATTAGCTGCT
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
917 x 3 = 2751
3617-2751 = 866
3617 bp
3,6 kb
O método de Sanger, 1975
Polimerização do DNA a ser sequenciado (molde)
na presença de:
DNA polimerase
primer
tampão
dNTPs (desóxinucleotídeo)
ddNTPs (didesóxinucleotídeo)
O que faria um nucleotídeo que,
ao invés da extremidade 3’OH,
tem uma extremidade 3’H?
http://www.youtube.com/watch?v=Mz4LSfecM4&feature=related (dideóxi)
Como acontece a síntese de
moléculas de DNA?
T
C
C
T
C
T
G
T
A
G
A G T
T
C A
A
C
AC T
T
C A
G
G
G T
T
A
G
C
A C C
C A
G
T
5’
3’
G
A
ATGCTTC
|||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
5’
T
T
C
T T
G
C
A
C A
T
A
G T
A
AC T
T
C A
G
G
G T
G
T
C C
AG
A C C
TC A G
5’
3’
G
A
ATGCTTC
|||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
C
5’
C
T
T
G
A
A GA T T
C
C CA
AC T
T
G
G T
A
G
T
G C
A
G
A C C
C A
G
T
5’
3’
G
A
ATGCTTCTG
||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
T
C
T C
C
T
AT G C T C
T
A T TT C A
T G
CA
C
G
A GC
T
G
G T
A
C A
G
T A G
A
C
C
A
GC
5’
3’
ATGCTTCTGGCAGATCT
|||||||||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
5’
A
T GAA
G
A
T
T
G
T
T
T
GA G C
C
T
A
T
T C A
GT G
C C
A
GC A T
C
G
C
AC
CC
5’
3’
ATGCTTCTGGCAGAT
|||||||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
5’
CC
T T
A
T GAA
G
A
G
C
T
A
C
T
T C AA G G
GT G
T
A
G
T
CA
C G C
C C
AC
5’
3’
ATGCTTCTGGCAGAT
|||||||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
T
3’
T
5’
A
5’
3’
T
T GAA CC
T
G
A
T
G
T
T
C A
G
C
T
G G
T AA
T
C
T
C G
G
T C
GC A
C
CA
AC
ATGCTTCTGGCAGAT
|||||||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
5’
5’
ATGCTTCTGGCAGATCTGAACAGTGTTACTGAT
ATGCTTCTGGCAGATCTGAACAGTGT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATAT
ATGCTTCTGGCAGATCTGAACAGTGTTACT
ATGCTTCTGGCAGATCTGAACAGT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCT
ATGCTTCTGGCAGATCT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCTGGCAGATCTGAACAGTGTT
ATGCTTCTGGCAGAT
||||||||||||||||||||||||||||||||||||||||
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA
3’
3’
População de
moléculas
Incorporação
aleatória do
didesóxi
Quantidade
precisa entre
didesóxi e
desóxi
5’
ATGCTTCT
ATGCTTCTGGCAGAT
ATGCTTCTGGCAGATCT
ATGCTTCTGGCAGATCTGAACAGT
ATGCTTCTGGCAGATCTGAACAGTGT
ATGCTTCTGGCAGATCTGAACAGTGTT
ATGCTTCTGGCAGATCTGAACAGTGTTACT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
• molde
• polimerase
• dNTPs
•ddGTPs
G
•ddATPs
A
•ddTTPs
T
•ddCTPs
C
ATGCTTCT
ATGCTTCTG
ATGCTTCTGG
ATGCTTCTGGC
ATGCTTCTGGCA
ATGCTTCTGGCAG
ATGCTTCTGGCAGA
ATGCTTCTGGCAGAT
ATGCTTCTGGCAGATC
ATGCTTCTGGCAGATCT
ATGCTTCTGGCAGATCTG
ATGCTTCTGGCAGATCTGA
ATGCTTCTGGCAGATCTGAA
ATGCTTCTGGCAGATCTGAAC
ATGCTTCTGGCAGATCTGAACA
ATGCTTCTGGCAGATCTGAACAG
ATGCTTCTGGCAGATCTGAACAGT
ATGCTTCTGGCAGATCTGAACAGTG
ATGCTTCTGGCAGATCTGAACAGTGT
ATGCTTCTGGCAGATCTGAACAGTGTT
ATGCTTCTGGCAGATCTGAACAGTGTTA
ATGCTTCTGGCAGATCTGAACAGTGTTAC
ATGCTTCTGGCAGATCTGAACAGTGTTACT
ATGCTTCTGGCAGATCTGAACAGTGTTACTG
ATGCTTCTGGCAGATCTGAACAGTGTTACTGA
ATGCTTCTGGCAGATCTGAACAGTGTTACTGAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATA
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTG
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGC
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
G A T C
ATGCTTCT
ATGCTTCTG
ATGCTTCTGG
ATGCTTCTGGC
ATGCTTCTGGCA
ATGCTTCTGGCAG
ATGCTTCTGGCAGA
ATGCTTCTGGCAGAT
ATGCTTCTGGCAGATC
ATGCTTCTGGCAGATCT
ATGCTTCTGGCAGATCTG
ATGCTTCTGGCAGATCTGA
ATGCTTCTGGCAGATCTGAA
ATGCTTCTGGCAGATCTGAAC
ATGCTTCTGGCAGATCTGAACA
ATGCTTCTGGCAGATCTGAACAG
ATGCTTCTGGCAGATCTGAACAGT
ATGCTTCTGGCAGATCTGAACAGTG
ATGCTTCTGGCAGATCTGAACAGTGT
ATGCTTCTGGCAGATCTGAACAGTGTT
ATGCTTCTGGCAGATCTGAACAGTGTTA
ATGCTTCTGGCAGATCTGAACAGTGTTAC
ATGCTTCTGGCAGATCTGAACAGTGTTACT
ATGCTTCTGGCAGATCTGAACAGTGTTACTG
ATGCTTCTGGCAGATCTGAACAGTGTTACTGA
ATGCTTCTGGCAGATCTGAACAGTGTTACTGAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATA
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATAT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTG
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGC
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
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 reação
de 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
Projetos Genoma e
Transcriptoma
A produção de
bibliotecas de DNA e cDNA
Prof. Dr. Francisco Prosdocimi
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 cDNA
DNA
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|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
>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
O formato GenBank
• http://www.ncbi.nlm.nih.gov
• Comandos LINUX