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Manipulating DNA and RNA. DNA hybridization PCR.

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Presentation on theme: "Manipulating DNA and RNA. DNA hybridization PCR."— Presentation transcript:

1 Manipulating DNA and RNA

2 DNA hybridization

3 PCR

4

5

6 PCR Movie

7 PCR Applications

8

9 PCR Applications Forensic Science

10 DNA Cloning

11 DNA cloning and amplification

12 Protein Production by DNA Cloning

13 Gene Mutation

14 Digestion and Destroy

15 Localization or Amplification of proteins by DNA Cloning

16 Detection of protein interactions by DNA Cloning

17

18 Yeast two-hybrid system

19 Identify Tumor Biomarkers by DNA Cloning Phage display

20 Northern and Southern Blots RNA and DNA Detection

21 Northern and Southern Blots

22 Microarray

23 SDS and b-mercaptoethanol for Electrophoresis

24 SDS PAGE I

25 SDS PAGE II

26 SDS PAGE III

27 Western Blot

28

29 Visualizing Cells

30 Resolving Power

31 Light Microscope

32 Interference between light waves

33 Resolution Calculation

34 Two ways to get contrast

35 Four Types of light microscopy Bright field, phase contrast, differential interference contrast, Dark-field microscopy

36 Fluorescence Microscope

37 Fluorescent Dyes

38 Fluorescent image Blue: DNA; Green: microtubules; Red: centrimere

39 Immunofluorescence

40 Immunostaining

41 Confocal Fluorescence Microscopy

42 The difference between conventional and confocal microscopes

43 3D reconstruction from confocal images

44 Transmission Electron microscopy (TEM, resolution 0.002 nm)

45 A root-tip cell under electromicroscopy

46 The scanning electron microscope (SEM)

47 Stereocillia from a hair cell

48 Live Cell Imaging

49 Green Fluorescence Protein (GFP)

50 Jellyfish and GFP Osamu Shimomura Noticed GFP in 1962

51 Dr. Douglas Prasher

52 Martin Chalfie

53 Sergey A. Lukyanov The Discovery of DsRed

54 Roger Y. Tsien

55 Track Molecular Motions

56 Passive Applications of GFP GFP-microtubules GFP-Actin CFP-Actin/YFP-Microtubules GFP-Actin

57

58 Spy on their Actions! FRET

59 The Principle of Fluorescence Resonance Energy Transfer (FRET) When the fluorophores are far apart: No FRET ExcitationEmission When fluorophores are close: FRET occurs ExcitationEmission FRET

60 FRET-Based Biosensors Calcium Ras and Rap1 Miyawaki, et al 1997, Nature Mochizuki, et al 2001, Nature Ting, et al 2001, PNAS Tyrosine Kinases

61 Design Strategy Weak FRET Phosphatase Strong FRET 433 nm 527 nm 433 nm 490 nm ECFP (1-227) SH2 (from c-Src) SubstrateEYFP Linker Src Activation

62 The Src reporter with CFP and YFP monomers ECFP (1-227) SH2 (from c-Src) SubstrateEYFP Linker A206K 0.5 0.35


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