1. Nucleic acid-based methods (I)

2. Structure of DNA

3. Complementality of DNA (high specificity in base paring)

4. Replication of DNA

5. Easy manipulation of DNA

6. Hybridization

7. Nucleic acid-based methods
Gene probing
Colony hybridization
Southern/Northern hybridization
Microarray
Polymerase Chain Reaction (PCR)
PCR
RT-PCR
Multiplex PCR
Nested PCR

8. Gene probes
Small pieces of DNA complementary to the target sequence of interest
Labeling options
Radioactive chemicals
Nonradioactive alternatives (DIG, biotin, or fluorescein)

10. Gene probe detection

11. Colony hybridization

12. Southern hybridization

13. Gel electrophoresis

14. Gel electrophoresis (I)

15. Gel electrophoresis (II)

16. DNA on a gel

17. Southern hybridization

18. An application of southern hybridization (forensic science)

19. Microbe Nucleic Acid Detection by DNA Microarrays or “Gene Chip” Technology
Generate/obtain DNA complimentary to genes (sequences) of interest;
1000s of different ones
Apply tiny quantities of each different one onto solid surfaces at defined positions
“gene chip” or “DNA microarray”
Isolate or amplify target NA of interest and label with a fluorescent probe
Apply sample NA to the “gene chip” surface
Sample NA binds to specific DNA probes on chip surface; wash away unbound NA
Detect bound DNA or RNA by fluorescence after laser excitation
Analyze hybridization data using imaging systems and computer software
Fluorescing Gene Chip or DNA Microarray

20. Polymerase Chain Reaction (PCR)

22. Primers and enzymes Primers Taq polymerase
Short nucleotides complementary to a target DNA
Upstream and downstream primers
Taq polymerase
Heat stable DNA-dependant DNA polymerase
Isolated from thermophilic bacteria (Thermus aquaticus)
Withstand temperature up to 98oC

24. Different PCR techniques
RT-PCR
Multiplex PCR
Nested PCR

28. Agarose Gel Electrophoresis
Separate nucleic acid fragments in an agarose gel
Resolves small DNA molecules: 0.1 to 50 kb
% agarose determines resolution of DNA size:
0.3% w/v: resolves 5 to 50 kb
2% w/v resolves 0.1 to 2 kb
Resolving large molecules (up to 500 kb) requires specialized methods
Pulse-field gel electrophoresis (PFGE)
DNA marker ladder
Specific
DNA
fragment

29. Example: RT-PCR and Oligoprobe Detection of Enteroviruses in Water

30. Real-Time PCR and Quantitative Fluorogenic Detection
Molecular beacon. Several 5' bases form base pairs with several 3' bases; reporter and quencher in close proximity.
If reporter is excited by light, its emission is absorbed by quencher & no fluorescence is detected.
Detection of PCR product by molecular beacon.
Beacon binds to PCR product and fluoresces when excited by the appropriate  of light.
[Fluorescence] proportional to [PCR product amplified]