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DNA Sequencing

DNA Sequencing. From Extraction to Information. The Process. Step 1: DNA Extraction Genomic DNA extraction from the organism ( bacteria ). Step 2: PCR Amplification Amplification of the DNA segment of interest ( 16S gene ). Step 3: DNA Sequencing

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DNA Sequencing

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  1. DNA Sequencing From Extraction to Information

  2. The Process • Step 1: DNA Extraction • Genomic DNA extraction from the organism (bacteria) • Step 2: PCR Amplification • Amplification of the DNA segment of interest (16S gene) • Step 3: DNA Sequencing • Sequencing of the PCR product (amplified16S gene)

  3. Step 1: DNA Extraction DNA Extraction DNA Cells with DNA

  4. Step 2: PCR Amplification PCR Product (Amplified Target Gene) Target Gene Target Gene PCR Amplification DNA

  5. Step 3: DNA Sequencing PCR product (Amplified Target Gene) Sequence of Target Gene DNA Sequencing AGCTGCTAAGCTTG AGCTTGCACAAGCT TAGCTTGCAAGCTT AGCTTGCAAGCTTG CAAGCTTGCAAGCT TGCAAGCTTGCAAG CTTGCAACGTTGCA AGCTTGCAAGCTTG AAGCTTGCAAGCTA

  6. Chapter 1:DNA Extraction

  7. The Cell and its Components DNA (1%) phospholipids (2%) 30% chemicals polysaccharides (2%) Ions, small molecules (4%) RNA (6%) 70% H2O proteins (15%)

  8. Three basic steps of DNA extraction • Disruption of cell and lysis • Removal of proteins and other biochemicals • Recovery of DNA

  9. Disruption of cell and lysis • Cells are broken down into components using a Lysis Buffer containing: • EDTA: disrupts cell membrane and inhibits DNases • SDS: denatures proteins and solubilizes cell membranes • Proteinase K: breaks down proteins • RNase A: breaks down RNA • Solution is incubated at 55ºC 1-3 hours (or overnight)

  10. Disruption of cell and lysis DNA RNA ions lipids proteins + + + + EDTA RNase A Proteinase K SDS EDTA

  11. Disruption of cell and lysis • After lysis, cell extract contains DNA, proteins, and other chemicals/biochemicals cell extract

  12. Removal of proteins and biochemicals • Solid phase binding (silica membrane) • Cell extract is applied to a silica membrane column • DNA binds to membrane • all other molecules flow through and are removed DNA bound to membrane Silica membrane centrifugation cell extract flow-thru

  13. Recovery of DNA • Elution of silica membrane: • a low-salt buffer or water is added to the membrane • bound DNA falls off of the membrane elution add water (or buffer) DNA bound to membrane centrifugation DNA in solution

  14. ReviewStep 1: DNA Extraction DNA Extraction DNA Cells with DNA

  15. Chapter 2: PCR Amplification

  16. Step 2: PCR Amplification PCR Product (Amplified Target Gene) Target Gene Target Gene PCR Amplification DNA

  17. Part I: DNA Polymerization

  18. DNA Building Block base 5’ P P P OCH2 O sugar 4’ 1’ H phoshpate groups H 3’ 2’ OH H deoxyribose nucleotide triphosphate (dNTP)

  19. DNA Polymerization: Basics A Existing DNA Strand O O G P O O C P O O T P Phosphodiester bond O H dNTP O T P P P O H O A P P P O H O C P P P O H

  20. DNA Polymerization • The synthesis of DNA requires: • DNA template • Primer: short oligonucleotide necessary for DNA polymerase to start • DNA polymerase: enzyme that constructs the DNA chain • deoxyribonucleotide triphosphates (dNTPs): building blocks of DNA A C C G G G A A G C C C C G G A T G A DNA polymerase A C G T DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  21. DNA Replication Review • Step 1: Denaturation: separation of the two strands of the DNA duplex • Gyrase pulls apart the strands creating a “replication bubble” • Helicase travels down DNA molecule, breaking the hydrogen bonds that hold the two strands together gyrase helicase helicase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G gyrase

  22. DNA Replication Review • Step 2: Annealing of primers to the DNA template strand • Primase synthesizes small complementary strands of RNA (“primers”) to the single strands of the DNA template primase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C G C C C C G primase G A T G A G C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  23. DNA Replication Review • Step 3: Extension of newly constructed complementary DNA molecules • DNA polymerase adds bases to the ends of the primers, constructing an exact copy of the template DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  24. DNA Replication Review • Another DNA Polymerase replaces the RNA primer with dNTPs • The final result: two copies of replicated DNA DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G G C C C C C C C C G G DNA polymerase G G A A T T G G A A G G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  25. P P P P P P P O O O O O O O O O O O O O O P P P P P P P P P P P P P P A C G T G T C P P P P P P O O O O O O O O O O O O O T C G A T A A G C T A T G C A A G C T O O O O O O O O O O O O O O O O O O O O O O O O O O O O P P P P P P P P P P P P P P Polymerization 1)DNA Template Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ 2)Primer 3)DNA Polymerase Mg2+ ions 4)dNTPs dNTPs 5)Mg2+ ions DNA Polymerase DNA Polymerase Primer Phosphodiester bond DNA Template

  26. Part II: PCR

  27. T A G T A C T C A G T A The Polymerase Chain Reaction • Polymerase Chain Reaction: cycling process consisting of the same 3 steps of DNA replication, with some differences: • temperature cycling removes the need for other enzymes (gyrase/helicase, or primase) • PCR uses pre-made oligonucleotide DNA primers DNA polymerase gyrase primase helicase

  28. The Polymerase Chain Reaction • During PCR, a thermocycler brings the reaction mix to 3 different temperatures analagous to the 3 steps of DNA replication • Denaturation (94˚C) of the DNA template by heat • Annealing (37˚-70˚C) of the primers to the template • Extension (72˚C) of the DNA strand by DNA polymerase • These steps are repeated for 25 to 30 cycles 94˚C 65˚C 72˚C denaturation annealing extension

  29. Thermocycler Program • Initial Denaturation: 94˚C 2 min • Start Cycle • Denaturation 94˚C 30 sec • Annealing 65˚C 30 sec • Extension 72˚C 30 sec • Repeat Cycle 29 times (total = 30 cycles) • Final Extension 72˚C 7 min • Hold 4˚C ∞

  30. Denaturation • Denaturation occurs at 94˚C • The high temperature is used to break down the hydrogen bonds that hold the two strands together 94˚C G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  31. T A G T A C T C A G T A Annealing • Annealing occurs at 37˚-70˚C • Oligonuclotide DNA primers anneal to their complementary sequences on the template strands • Annealing temperature depends on the melting temperature (Tm) of the primer (dependent on base composition) 94˚C 65˚C G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  32. Extension • Extension occurs at 72˚C • DNA polymerase attaches to the primers and extends the new DNA strand • The 3 steps (denaturation, annealing, and extension) are repeated for another 24 to 29 cycles 65˚C 72˚C DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C DNA polymerase T C A G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  33. Target Sequence • A desired target sequence is identified • To isolate the target sequence, primers that flank the region must be constructed • The DNA segment that is then amplified contains the region of interest Template DNA Forward Primer Reverse Primer Target Sequence of interest PCR Product

  34. PCR: Cycle 1 Denaturation Extension Annealing DNA Copies 4 Target Copies 0 Target Sequence of interest

  35. PCR: Cycle 2 Denaturation Extension Annealing DNA Copies 8 Target Copies 2

  36. PCR: Cycle 3 Denaturation Extension Annealing DNA Copies 16 Target Copies 8

  37. PCR: Cycle 4 Denaturation Extension Annealing DNA Copies 32 Target Copies 22

  38. PCR: Cycle 5 Denaturation Extension Annealing DNA Copies 64 Target Copies 52

  39. PCR Amplification: First 10 cycles

  40. PCR Amplification: First 15 cycles

  41. PCR Amplification: After 30 cycles

  42. PCR Amplification: After 30 cycles

  43. ReviewStep 1: DNA Extraction DNA Extraction DNA Cells with DNA

  44. ReviewStep 2: PCR Amplification PCR Product (Amplified Target Gene) Target Gene Target Gene PCR Amplification DNA

  45. Chapter 3:DNA Sequencing

  46. Nucleotides BASE BASE OCH2 P P P OCH2 P P P O O H H H H OH H OH OH deoxyribose NTP (dNTP) (Makes up DNA) ribose NTP (NTP) (Makes up RNA) BASE OCH2 P P P O H H H H dideoxyribose NTP (ddNTP)

  47. DNA Sequencing • Dideoxy method of DNA sequencing (Sanger Method) • Single-stranded DNA to be sequenced serves as a template strand for DNA synthesis • single primer is used for DNA synthesis initiation • use of dNTPs along with labeled ddNTPs BASE BASE OCH2 OCH2 P P P P P P O O H H H H OH H H H dNTP ddNTP

  48. DNA Polymerization using ddNTPs A A O O O G P O G P O O O C P O C P O O O T P O C P O H O H O T P P P O T P P P O H H O A P P P O A P P P O H O H O C P P P Chain Termination O H

  49. Sequence Reaction • BigDye Terminator v3.1 Sequencing: • a Dye Terminator Cycle Sequencing Master Mix is used for sequencing reaction. Components include: • DNA polymerase I, Mg2+, buffer • dNTPs in ample quantities: • (dATP, dTTP, dCTP, dGTP) • ddNTPs in limited quantities, each labeled with a “tag” that fluoresces a different “color”: • (ddATP, ddTTP, ddCTP, ddGTP)

  50. The Polymerase Chain Reaction • PCR makes use of a thermocycler to bring the reaction mix to three different temperatures • Denaturation (94˚C) of the DNA template by heat • Annealing (37˚-70˚C) of the primers to the template • Extension (72˚C) of the DNA strand by DNA polymerase • These steps are repeated for 25 to 30 cycles 94˚C 65˚C 72˚C denaturation annealing extension

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