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Sanger or Dideoxy DNA Sequencing

Sanger or Dideoxy DNA Sequencing. Components for the Chain Termination Method : DNA fragment for sequencing Primers dNTPs ddNTPs. Sanger or Dideoxy DNA Sequencing. What direction does the DNA polymerase elongate the DNA fragment?. Sanger or Dideoxy DNA Sequencing.

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Sanger or Dideoxy DNA Sequencing

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  1. Sanger or Dideoxy DNA Sequencing Components for the Chain Termination Method: • DNA fragment for sequencing • Primers • dNTPs • ddNTPs

  2. Sanger or Dideoxy DNA Sequencing What direction does the DNA polymerase elongate the DNA fragment?

  3. Sanger or Dideoxy DNA Sequencing Migration smaller is faster through the gel (bottom smallest) Each peak represents 1 base

  4. Pyrosequencing Based on DNA synthesis Template DNA is immobilized Peak area represents base number http://www.pyrosequencing.com/DynPage.aspx?id=7454

  5. Pyrosequencing Potential Problem: DNA polymerase and luciferase compete for the same dATP substrate Solution: Deoxyadenosine alpha-thio triphosphate (dATPαS) is efficiently used by DNA polymerase but not recognized by luciferase

  6. Pyrosequencing Requirement: Generate ATP from polymerase generated pyrophosphate Solution: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate

  7. Pyrosequencing Potential Problem: Unincorporated dNTPs can react in the next cycle producing false positives Solution: The nucleotide degrading enzyme apyrase degrades unincorporated dNTPs between cycles

  8. Gene Amplification by Polymerase Chain Reaction (PCR) Gene specific amplification without purification Amplification via temperature cycling Special Taq (Thermus aquaticus) DNA polymerase Detection of Helicobacter pylori, Borrelia burgdorferi, as well as hepatitis, HIV, and West Nile virus

  9. Restriction Enzyme Recognition Sites Restrictive endonucleases recognize 4-8 bp sequences within the DNA and cleave at a specific site DNA strands that are cut in a symmetric fashion are palindromic Blunt and Sticky DNA Cuts

  10. Molecular Cloning by Recombinant DNA Technology • Cut by restriction enzymes • Anneal an uncatalyzed reaction • Re-close by DNA ligase

  11. Screening for Colonies Containing the Plasmid with the Insert

  12. Size-Specific Cloning Vectors

  13. Recombinant Protein Products

  14. Generating a Restriction Map Where are the restriction sites located on the 20 kb fragment that would generate this map? . . .

  15. Transcript Analysis via Microarray/DNA Chip How to examine gene expression changes with a given treatment mRNA ↓ cDNA fluorecent labeling ↓ cDNA/oligonucleotide Hybridization ↓ Slide scanning ↓ Data analysis

  16. Differential Arabidopsis Gene Expression with GB03 Exposure RNA Extraction cDNA Synthesis Label Hybridize Wash Scan Block 8 Full Image

  17. cDNA Synthesis and Labeling Amino Allyl-dUTP Cy5 Dye

  18. Microarray DataAnalysis

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