Biologia Molecular, revisão do conteúdo Prof Francisco Prosdocimi.
Transcript of Biologia Molecular, revisão do conteúdo Prof Francisco Prosdocimi.
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)
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
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||||||||||
5’3’
ATGCTTCATGCTTCTGGCAGATGGCAGATT5’ 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
TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAATACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA|||||||||||||||
5’3’
ATGCTTCATGCTTCTGGCAGATGGCAGATT5’ 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
ATGCTTCATGCTTCTGGCAGATGGCAGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATGGCAGATCTGAACAGTGTTACTGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTGGCAGATCTGAACAGTGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTGGCAGATCTGAACAGTGTTACTGATATTGCTTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGGCAGATCTGAACAGTGTTACTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGGCAGATCTGAACAGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTGGCAGATCTGAACAGTGTTACTGATATTGCTTATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGGCAGATCTGAACAGTGTTACTGATATTT
ATGCTTCATGCTTCTT
ATGCTTCATGCTTCTGGCAGATCTGGCAGATCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTGGCAGATCTGAACAGTGTTT
G A T C
• moldemolde• polimerasepolimerase• dNTPsdNTPs
•ddGTPsddGTPs •ddATPsddATPs •ddTTPsddTTPs •ddCTPsddCTPs
ATGCTTCATGCTTCTGGCAGATGGCAGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATGGCAGATCTGAACAGTGTTACTGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTGGCAGATCTGAACAGTGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTGGCAGATCTGAACAGTGTTACTGATATTGCTTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGGCAGATCTGAACAGTGTTACTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGGCAGATCTGAACAGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGGCAGATCTGAACAGTGTTACTGATATTT
ATGCTTCATGCTTCTT
ATGCTTCATGCTTCTGGCAGATCTGGCAGATCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTGGCAGATCTGAACAGTGTTT
ATGCTTCATGCTTCTTGGATGCTTCATGCTTCTGTGGG
ATGCTTCATGCTTCTGGCATGGCAGG
ATGCTTCATGCTTCTGGCAGATCTTGGCAGATCTGG
ATGCTTCATGCTTCTGGCAGATCTGAACATGGCAGATCTGAACAGG
ATGCTTCATGCTTCTGGCAGATCTGAACAGTTGGCAGATCTGAACAGTGG
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTTGGCAGATCTGAACAGTGTTACTGG
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTTGGCAGATCTGAACAGTGTTACTGATATTGG
ATGCTTCATGCTTCTGGTGGCC
ATGCTTCATGCTTCTGGCAGATTGGCAGATCC
ATGCTTCATGCTTCTGGCAGATCTGAATGGCAGATCTGAACC
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTATGGCAGATCTGAACAGTGTTACC
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGTGGCAGATCTGAACAGTGTTACTGATATTGCC
ATGCTTCATGCTTCTGGCTGGCAA
ATGCTTCATGCTTCTGGCAGTGGCAGAA
ATGCTTCATGCTTCTGGCAGATCTGTGGCAGATCTGAAATGCTTCATGCTTCTGGCAGATCTGATGGCAGATCTGAAA
ATGCTTCATGCTTCTGGCAGATCTGAACTGGCAGATCTGAACAA
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTTGGCAGATCTGAACAGTGTTAA
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGTGGCAGATCTGAACAGTGTTACTGAA
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATTGGCAGATCTGAACAGTGTTACTGATAA
G A T C
ATGCTTCATGCTTCTGGCAGATGGCAGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATGGCAGATCTGAACAGTGTTACTGATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTGGCAGATCTGAACAGTGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTGGCAGATCTGAACAGTGTTACTGATATTGCTTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATGGCAGATCTGAACAGTGTTACTGATATT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGGCAGATCTGAACAGTGTTACTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGGCAGATCTGAACAGTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGGCAGATCTGAACAGTGTTACTGATATTT
ATGCTTCATGCTTCTT
ATGCTTCATGCTTCTGGCAGATCTGGCAGATCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTACTGATATTGCTTTGGCAGATCTGAACAGTGTTACTGATATTGCTT
ATGCTTCATGCTTCTGGCAGATCTGAACAGTGTTGGCAGATCTGAACAGTGTTT
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