manipulating dna and rna. dna hybridization pcr
TRANSCRIPT
• Manipulating DNA and RNA
DNA hybridization
PCR
PCR Movie
PCR Applications
PCR Applications
PCR Applications Forensic Science
DNA Cloning
DNA cloning and amplification
Protein Production by DNA Cloning
Gene Mutation
Gene Mutation
Digestion and Destroy
Localization or Amplification of proteins by DNA Cloning
Detection of protein interactions by DNA Cloning
Detection of protein interactions by DNA Cloning
Detection of protein interactions by DNA CloningYeast two-hybrid system
Identify Tumor Biomarkers by DNA CloningPhage display
Northern and Southern BlotsRNA and DNA Detection
Northern and Southern Blots
Microarray
SDS and b-mercaptoethanol for Electrophoresis
SDS PAGE I
SDS PAGE II
SDS PAGE III
Western Blot
Western Blot
• Visualizing Cells
Resolving Power
Light Microscope
Interference between light waves
Resolution Calculation
Two ways to get contrast
Four Types of light microscopyBright field, phase contrast, differential
interference contrast, Dark-field microscopy
Fluorescence Microscope
Fluorescent Dyes
Fluorescent imageBlue: DNA; Green: microtubules; Red: centrimere
Immunofluorescence
Immunostaining
Confocal Fluorescence Microscopy
The difference between conventional and confocal microscopes
3D reconstruction from confocal images
Transmission Electron microscopy (TEM, resolution 0.002 nm)
A root-tip cell under electromicroscopy
The scanning electron microscope (SEM)
Stereocillia from a hair cell
Live Cell Imaging
Green Fluorescence Protein (GFP)
Jellyfish and GFP
Osamu ShimomuraNoticed GFP in 1962
Dr. Douglas Prasher
Martin Chalfie
Sergey A. Lukyanov
The Discovery of DsRed
Roger Y. Tsien
Track Molecular Motions
Passive Applications of GFP
GFP-microtubules GFP-Actin
CFP-Actin/YFP-Microtubules GFP-Actin
Spy on their Actions!
FRET
The Principle of Fluorescence Resonance Energy Transfer (FRET)
When the fluorophores are far apart: No FRET
Excitation Emission
When fluorophores are close: FRET occurs
Excitation Emission
FRET
FRET-Based BiosensorsCalcium Ras and Rap1
Miyawaki, et al 1997, Nature
Mochizuki, et al 2001, Nature
Ting, et al 2001, PNAS
Tyrosine Kinases
Design Strategy
Weak FRET
Phosphatase
Strong FRET
433 nm
527 nm
433 nm
490 nm
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
Src Activation
The Src reporter with CFP and YFP monomers
ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker
A206K A206K
0.5
0.35