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

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. cDNA fluorecent labeling cDNA/oligonucleotide
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 Data Analysis