Manipulating DNA and RNA. DNA hybridization PCR.

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Manipulating DNA and RNA

Transcript of Manipulating DNA and RNA. DNA hybridization PCR.

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• Manipulating DNA and RNA

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DNA hybridization

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PCR

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PCR Movie

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PCR Applications

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PCR Applications

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PCR Applications Forensic Science

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DNA Cloning

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DNA cloning and amplification

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Protein Production by DNA Cloning

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Gene Mutation

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Gene Mutation

Digestion and Destroy

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Localization or Amplification of proteins by DNA Cloning

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Detection of protein interactions by DNA Cloning

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Detection of protein interactions by DNA Cloning

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Detection of protein interactions by DNA CloningYeast two-hybrid system

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Identify Tumor Biomarkers by DNA CloningPhage display

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Northern and Southern BlotsRNA and DNA Detection

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Northern and Southern Blots

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Microarray

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SDS and b-mercaptoethanol for Electrophoresis

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SDS PAGE I

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SDS PAGE II

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SDS PAGE III

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Western Blot

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Western Blot

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• Visualizing Cells

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Resolving Power

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Light Microscope

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Interference between light waves

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Resolution Calculation

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Two ways to get contrast

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Four Types of light microscopyBright field, phase contrast, differential

interference contrast, Dark-field microscopy

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Fluorescence Microscope

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Fluorescent Dyes

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Fluorescent imageBlue: DNA; Green: microtubules; Red: centrimere

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Immunofluorescence

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Immunostaining

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Confocal Fluorescence Microscopy

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The difference between conventional and confocal microscopes

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3D reconstruction from confocal images

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Transmission Electron microscopy (TEM, resolution 0.002 nm)

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A root-tip cell under electromicroscopy

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The scanning electron microscope (SEM)

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Stereocillia from a hair cell

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Live Cell Imaging

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Green Fluorescence Protein (GFP)

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Jellyfish and GFP

Osamu ShimomuraNoticed GFP in 1962

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Dr. Douglas Prasher

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Martin Chalfie

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Sergey A. Lukyanov

The Discovery of DsRed

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Roger Y. Tsien

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Track Molecular Motions

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Passive Applications of GFP

GFP-microtubules GFP-Actin

CFP-Actin/YFP-Microtubules GFP-Actin

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Spy on their Actions!

FRET

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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

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FRET-Based BiosensorsCalcium Ras and Rap1

Miyawaki, et al 1997, Nature

Mochizuki, et al 2001, Nature

Ting, et al 2001, PNAS

Tyrosine Kinases

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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

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The Src reporter with CFP and YFP monomers

ECFP(1-227) SH2(from c-Src) Substrate EYFPLinker

A206K A206K

0.5

0.35