The document describes the Sanger method for DNA sequencing, which was developed in 1977. The method uses DNA polymerase and dideoxynucleotides to terminate DNA strand elongation at random positions, generating DNA fragments of different lengths that can be used to determine the DNA sequence. The sequence is read by comparing the fragment lengths generated from reactions with different labeled dideoxynucleotides.
2. 1977: Walter Gilbert e Frederick Sanger seqüenciamento de DNA Nobel 1980 “ pelas contribui ções na determinação de seqüências de ácidos nucleicos ”
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4. Procedimento enzimático (Sanger, 1977) A posição das bases é determinada pelo tamanho dos fragmentos obtidos através de reações de polimerização na presença de dideoxinucleotídeos .
5. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||| 5’ 3’ ATGCTTC 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C
6. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC 5’ 3’
7. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TG 5’
8. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCT 5’
9. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCT 5’
10. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACA 5’
11. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTT 5’
12. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTTACTG 5’
13. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTTACTGAT 5’
14. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTTACTGAT 5’
15. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||||||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT 5’
16. Polimerização de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT 5’
17. Dideoxinucleotídeo di deoxinucleotídeo O - O - P - P - P - O - CH 2 - O - O O O BASE - O - - O - H 3' 2' H O - O - P - P - P - O - CH 2 - O - O O O BASE - O - - O - H 3' 2' H deoxinucleotídeo O - O - P - P - P - O - CH 2 - O - O O O BASE - O - - O - OH 3' 2' H O - O - P - P - P - O - CH 2 - O - O O O BASE - O - - O - 3' 2' H
19. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||| 5’ 3’ ATGCTTC 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C
20. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC 5’ 3’
21. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TG 5’
22. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCT 5’
23. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGATCT 5’
24. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGA T 5’
25. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGA T 5’
26. Polimerização de DNA o dideoxi TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA ||||||||||||||| 5’ 3’ A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C ATGCTTC TGGCAGA T 5’
27. Seqüenciamento de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||||||||||||| 5’ 3’ ATGCTTC TGGCAGA T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT ATGCTTC TGGCAGATCTGAACAGTGT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGA T 5’ 3’ ATGCTTC TGGCAGATCTGAACAGTG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATA T ATGCTTC TGGCAGATCTGAACAGTGTTAC T ATGCTTC TGGCAGATCTGAACAG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATAT T ATGCTTC T ATGCTTC TGGCAGATC T
28. Seqüenciamento de DNA TACGAAGACCGTCTAGACTTGTCACAATGACTATAACGAA |||||||||||||||||||||||||||||||||||||||| 5’ 3’ ATGCTTC TGGCAGA T ATGCTTC TGGCAGATCTGAACAGTGTTACTGA T ATGCTTC TGGCAGATCTGAACAGTG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATA T ATGCTTC TGGCAGATCTGAACAGTGTTAC T ATGCTTC TGGCAGATCTGAACAG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATAT T ATGCTTC T ATGCTTC TGGCAGATC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT ATGCTTC TGGCAGATCTGAACAGTGT T
29. Seqüenciamento de DNA ATGCTTC TGGCAGA T ATGCTTC TGGCAGATCTGAACAGTGTTACTGA T ATGCTTC TGGCAGATCTGAACAGTG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATA T ATGCTTC TGGCAGATCTGAACAGTGTTAC T ATGCTTC TGGCAGATCTGAACAG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATAT T ATGCTTC T ATGCTTC TGGCAGATC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT ATGCTTC TGGCAGATCTGAACAGTGT T
32. Seqüenciamento de DNA ATGCTTC TGGCAGA T ATGCTTC TGGCAGATCTGAACAGTGTTACTGA T ATGCTTC TGGCAGATCTGAACAGTG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATA T ATGCTTC TGGCAGATCTGAACAGTGTTAC T ATGCTTC TGGCAGATCTGAACAG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATAT T ATGCTTC T ATGCTTC TGGCAGATC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT ATGCTTC TGGCAGATCTGAACAGTGT T ATGCTTC T G ATGCTTC TG G ATGCTTC TGGCA G ATGCTTC TGGCAGATCT G ATGCTTC TGGCAGATCTGAACA G ATGCTTC TGGCAGATCTGAACAGT G ATGCTTC TGGCAGATCTGAACAGTGTTACT G ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATT G ATGCTTC TGG C ATGCTTC TGGCAGAT C ATGCTTC TGGCAGATCTGAA C ATGCTTC TGGCAGATCTGAACAGTGTTA C ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTG C ATGCTTC TGGC A ATGCTTC TGGCAG A ATGCTTC TGGCAGATCTG A ATGCTTC TGGCAGATCTGA A ATGCTTC TGGCAGATCTGAAC A ATGCTTC TGGCAGATCTGAACAGTGTT A ATGCTTC TGGCAGATCTGAACAGTGTTACTG A ATGCTTC TGGCAGATCTGAACAGTGTTACTGAT A G A T C
33. Seqüenciamento de DNA ATGCTTC TGGCAGA T ATGCTTC TGGCAGATCTGAACAGTGTTACTGA T ATGCTTC TGGCAGATCTGAACAGTG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCT T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATA T ATGCTTC TGGCAGATCTGAACAGTGTTAC T ATGCTTC TGGCAGATCTGAACAG T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATAT T ATGCTTC T ATGCTTC TGGCAGATC T ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTGCTT ATGCTTC TGGCAGATCTGAACAGTGT T ATGCTTC T G ATGCTTC TG G ATGCTTC TGGCA G ATGCTTC TGGCAGATCT G ATGCTTC TGGCAGATCTGAACA G ATGCTTC TGGCAGATCTGAACAGT G ATGCTTC TGGCAGATCTGAACAGTGTTACT G ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATT G ATGCTTC TGG C ATGCTTC TGGCAGAT C ATGCTTC TGGCAGATCTGAA C ATGCTTC TGGCAGATCTGAACAGTGTTA C ATGCTTC TGGCAGATCTGAACAGTGTTACTGATATTG C ATGCTTC TGGC A ATGCTTC TGGCAG A ATGCTTC TGGCAGATCTG A ATGCTTC TGGCAGATCTGA A ATGCTTC TGGCAGATCTGAAC A ATGCTTC TGGCAGATCTGAACAGTGTT A ATGCTTC TGGCAGATCTGAACAGTGTTACTG A ATGCTTC TGGCAGATCTGAACAGTGTTACTGAT A
42. Procedimento químico (Maxam & Gilbert, 1977) separação das fitas DNA marcado na extremidade 5' reações parciais de clivagens específicas 5' 5' 5' T G A C G C T G A T gel desnaturante de poliacrilamida (uréia 8 M) G A+G T+C C T G A C G C T G A T 5' 8 pb 5 pb 2 pb e l e t r o f o r e s e
47. Genome Sequencer 20 System Roche Applied Science 454 Life Sciences Genome sequencing in microfabricated high-density picolitre reactors. Nature 437 , 376-380 (15 September 2005) The apparatus (…) is able to sequence 25 million bases, at 99% or better accuracy, in one 4-hour run. (…) shotgun sequencing and de novo assembly of the Mycoplasma genitalium [580 Mb] genome with 96% coverage at 99.96% accuracy in one run of the machine. http://dx.doi.org/10.1038/nature03959
48. Pirosseqüenciamento ATP + Luciferina + O 2 AMP + PPi + Oxiluciferina + CO 2 + Luz Luciferase PPi + APS SO 4 + ATP ATP Sulforilase DNA n + Nucleot ídeo DNA n+1 + PPi Polimerase