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DNA sequencing: Importance

DNA sequencing: Importance. Basic blueprint for life; Aesthetics. Gene and protein. Function Structure Evolution Genome-based diseases- “inborn errors of metabolism.” Genetic disorders Genetic predispositions to infection Diagnostics Therapies. Maxam-Gilbert

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DNA sequencing: Importance

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  1. DNA sequencing: Importance • Basic blueprint for life; Aesthetics. • Gene and protein. • Function • Structure • Evolution • Genome-based diseases- “inborn errors of metabolism.” • Genetic disorders • Genetic predispositions to infection • Diagnostics • Therapies

  2. Maxam-Gilbert base modification by general and specific chemicals. depurination or depyrimidination. single-strand excision. not amenable to automation Sanger DNA replication. substitution of substrate with chain-terminator chemical. more efficient automation?? DNA sequencing methodologies

  3. Maxam-Gilbert chemical method

  4. Sanger dideoxynucleotides versus “bio” based methods

  5. DNA biochemistry

  6. Sequence Masters • Fred Sanger, 1958 • Was originally a protein chemist • Made his first mark in sequencing proteins • Made his second mark in sequencing RNA • 1980 dideoxy sequencing

  7. The Sanger Method • Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA • Requires DNA polymerase I.. Why? • Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker) • Uses 32P-deoxynucleoside triphosphates

  8. Sanger Method Sequencing Gel

  9. O O O O O O P P P OH OH OH DNA sequencing: biochemistry 5’ purine or pyrimidine N HO C O purine or pyrimidine O N C O O O P OH 3’ OH

  10. O O O O O O P P P OH OH OH DNA sequencing: Sanger dideoxy method I purine or pyrimidine N HO C O dideoxyribonucleoside triphosphate (ddNTP) H

  11. O O O O O O P P P OH OH OH DNA sequencing: Sanger II purine or pyrimidine N HO C O purine or pyrimidine O chain termination method N C O O O P OH H

  12. DNA sequencing: Chemistry

  13. DNA sequencing: Chemistry template + primers + polymerase +label at? 1 dCTP dTTP dGTP dATP ddATP* 2 dCTP dTTP dGTP dATP ddGTP* 3 dCTP dTTP dGTP dATP ddTTP* 4 dCTP dTTP dGTP dATP ddCTP* electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T extension

  14. Manual radioactive sequencing

  15. DNA sequencing: Chemistry template + polymerase + 1 dCTP dTTP dGTP dATP ddATP primer 2 dCTP dTTP dGTP dATP ddGTP primer 3 dCTP dTTP dGTP dATP ddTTP primer 4 dCTP dTTP dGTP dATP ddCTP primer electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T extension

  16. Semi-automated fluorescent DNA sequencing • Fred Sanger et. al., 1977. • Walter Gilbert et. al., 1977. • Leroy Hood et. al. 1986. • Applied Biosystems, Inc. • DuPont Company.

  17. DNA sequencing: upgrade, second iteration, terminator-label • Disadvantages of primer-labels: • four reactions • tedious • limited to certain regions, custom oligos or • limited to cloned inserts behind ‘universal’ priming sites. • Advantages: • Solution: • fluorescent dye terminators

  18. DNA sequencing: Chemistry template + polymerase + dCTP dTTP dGTP dATP ddATP ddGTP ddTTP ddCTP electrophoresis A•T G•C A•T T•A C•G T•A G•C G•C A•T G•C T•A T•A C•G T•A G•C A•T extension

  19. DNA sequencing: Chemistry

  20. Sequence Masters • Walter Gilbert • Harvard physicist • Knew James Watson • Became intrigued with the biological side • Became a biophysicist • Allan Maxam

  21. The Maxam-Gilbert Technique • Principle - Chemical Degradation of Purines • Purines (A, G) damaged by dimethylsulfate • Methylation of base • Heat releases base • Alkali cleaves G • Dilute acid cleave A>G

  22. The Maxam-Gilbert Technique • Principle – Chemical Degradation of Pyrimidines • Pyrimidines (C, T) are damaged by hydrazine • Piperidine cleaves the backbone • 2 M NaCl inhibits the reaction with T

  23. The Maxam-Gilbert Method

  24. Sanger Method Enzymatic Requires DNA synthesis Termination of chain elongation Maxam Gilbert Method Chemical Requires DNA Requires long stretches of DNA Breaks DNA at different nucleotides Comparison

  25. Sequencing Gives: • The letters in a sentence • Remember Prions? • Short sequence in genomes • Single nucleotide change in alleles • Valine - Valine = not susceptible to BSE • Methionine - Valine = at risk • Methionine-methionine = watch out! • How can we genetically screen for single nucleotide differences?

  26. Applications DNA sequencing • Whole genome analysis • Comparative genomics • Applications to subfields

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