Advanced Nucleic Acid-Based Methods in Molecular Biology

1. Nucleic acid-based methods (I)

2. Structure of DNA (I)

3. Structure of DNA (II)

4. Structure of DNA (III)

5. Replication of DNA

6. Denaturation and Reannealing

7. Hybridization

8. Nucleic-acid based methods • Gene probing • Colony hybridization • Southern/Northern hybridization • Microarray • Polymerase Chain Reaction (PCR) • PCR • RT-PCR • Multiplex PCR • Nested PCR • Restriction Fragment Length Polymorphism (RFLP) analysis

9. 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

12. Colony hybridization

13. Gel electrophoresis

14. Gel electrophoresis (I)

15. Gel electrophoresis (II)

16. DNA on a gel

17. Southern hybridization (I)

18. Southern hybridization (II)

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. Gene Chip

21. Polymerase Chain Reaction (PCR)

23. Primers and enzymes • Primers • 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 98 oC

25. Nucleic Acid Amplification - PCR

26. Different PCR techniques • PCR • RT-PCR • Multiplex PCR • Nested PCR

30. 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

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

32. 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]