生物科技研究方法
Tools for analyzing gene expression
Reference: fundamental molecular biology,
Lizabeth Allison, 2007
蔡士彰
Central dogma
DNA
RNA
protein
Level
Methods
Transcription
Northern blot
In situ hybridization
RNase protection assay
Reverse transcriptase PCR (RT-PCR)
Western blot
ELISA
Translation
DNA-protein
interaction
EMSA,
DNase I footprinting,
Chromatin immunoprecipitation (ChIP) assay
Protein-protein Pull down assay
interaction
Yeast two hybrid assay
Coimmunoprecipition
Fluorescence resonance energy trnsfer (FRET)
RNA DETECTION & ANALYSIS
**Steady-state levels of RNA**
** RNA levels that exist as a result of **
Transcription (+) versus degradation (-)
METHODS:
•Northern blotting
•RNase protection assay
•RT-PCR
•Microarrays
RNase Protection Assay
+1 (start site)
RPA
+50
-100
T7 or SP6 pr omoter to
make antisense RNA
HindII Isite
used to li near ize
plasmid
tr anscri ption bufferRNA
,
polymer ase ,
NTP's + [ -32P]UTP to make radiolabele d
antisense RNA pr obe
-100
+1
3'
+50
5'
hybr idize pr obe to purified
mRNA
Add RNase
-100
3'
+1
+50
5'
5'
digest wi th RNaseT1 and RNase A
(cleaves single-stranded
RNAs)
+1
AAAAAAA
3' mRN A
+50
RNA duplex protected
creates a RNA RNA hybr id
den atur e an d analyze on a
denatur ingpolyacr yl ami degel e
G A T C
>150 nt
50 nt
RT-PCR
mRNA
Reverse transcription
PCR
Genome-wide comparison of mRNA expression between tumor and normal cells
Microarrays
Massively parallel analysis of gene expression
• screen an entire genome at once
• find not only individual genes that differ,
but groups of genes that differ.
• find relative expression level differences
Effective for identify regulatory networks
Reporter Assay
Transfect
embryo or ES cells
DNA transfection methods
1. Calcium phosphate-DNA
Coprecipitation method
2. DEAE-Dextran
3. Electroporation
4. Micro injection
5. Liposome
Analysis of expression pattern
in whole organism
Direct enzymatic activity assay
of cell lysate
The major approaches of DNA delivery
Physical delivery
Chemical delivery
Viral vectors
Bactofection (bacterial vectors)
Chemical delivery
1. Calcium-phosphate mediated transfection
2. polyplexes: eg. DEAE-dextran
3. Cationic liposomes: eg. Lipofectin, lipofectamine, lipofectase….
Physical delivery: electroporation, microinjection, gene
gun (microballistics)
Electroporation:
Microinjection:
Reporter genes
X-gal
IPTG: an inducer of β-galactosidase. This compound is used as a molecular
mimic of allolactose
Lactose is a disaccharide that consists of β-D-__________
and β-D-____________ molecules bonded through a β1-4
glycosidic linkage.
X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo4-chloro-3-hydroxyindole. 5-bromo-4-chloro-3-hydroxyindole then
is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue
product.
Luciferase is a generic name for enzymes commonly used in nature for
bioluminescence. The name itself is derived from Lucifer, which means lightbearer. The most famous one is firefly luciferase from the firefly Photinus pyralis.
In luminescent reactions, light is produced by the oxidation of a luciferin (a
pigment), sometimes involving Adenosine triphosphate (ATP).
The rates of this reaction between luciferin and oxygen are extremely slow until
they are catalyzed by luciferase
The reaction takes place in two steps:
luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light
The reaction is very energy efficient: nearly all of the energy input into the reaction
is transformed into light. As a comparison, the incandescent light bulb loses about
90% of its energy to heat.
luciferase (luc) systems
firefly species Photinus pyralis
Expressed luciferase catalyses
oxidation of compounds called luciferans
( ATP-dependent process)
These compounds emit
fluorescense
luminometer measurement
Mice are injected
with LUC+ salmonellas.
Sensitive digital cameras
allow non-invasive detection.
For GT vectors pictures
look the same
Chloramphenicol
acetyltransferase is a
bacterial enzyme that
detoxifies the antibiotic
chloramphenicol.
It is responsible for
chloramphenicol
resistance in bacteria.
This enzyme covalently
attaches an acetyl group
from acetyl-CoA to the
chloramphenicol molecule
so that it is unable to bind
to the ribosome.
Reporter gene systems
chloramphenicol acetyl transferase (CAT)
CAT is a bacterial enzyme that
catalyzes the transfer of acetyl
groups from acetylcoenzyme A to the antibiotic
chloramphenicol.
(chloramphenicol deactivation)
thin-layer chromatographic sheet
Chloramphenicol is
radiolabelled
Green fluorescent protein (GFP)
autofluorescent protein from Pacific Northwest
jellyfish Aequorea victoria
GFP is an extremely stable protein
of 238 amino acids with unique post-translationally
created and covalently-attached chromophore from
oxidised residues 65-67, Ser-Tyr-Gly
ultraviolet light causes GFP
to autofluoresce
In a bright green color
Jellyfish do nothing with UV,
The activate GFP by aequorin
(Ca++ activated,
biolumuniscent helper)
GFP expression is harmless
for cells and animals
GFP construct could be used for construct tracking in living organism
GFP labelled image of a human tumor.
Vessel on the tumor surface
are visible in black
Level
Methods
Transcription
Northern blot
In situ hybridization
RNase protection assay
Reverse transcriptase PCR (RT-PCR)
Western blot
ELISA
Translation
DNA-protein
interaction
EMSA,
DNase I footprinting,
Chromatin immunoprecipitation (ChIP) assay
Protein-protein Pull down assay
interaction
Yeast two hybrid assay
Coimmunoprecipition
Fluorescence resonance energy trnsfer (FRET)
Level
Methods
Transcription
Northern blot
In situ hybridization
RNase protection assay
Reverse transcriptase PCR (RT-PCR)
Western blot
ELISA
Translation
DNA-protein
interaction
EMSA,
DNase I footprinting,
Chromatin immunoprecipitation (ChIP) assay
Protein-protein Pull down assay
interaction
Yeast two hybrid assay
Coimmunoprecipition
Fluorescence resonance energy trnsfer (FRET)
Electrophoretic-Mobility Shift Assay (EMSA)
DNA binding site
20-30 nt probe
+
DNA binding protein
Protein/DNA complex
Electrophoretic-Mobility Shift Assay
Incubate protein and DNA probe
Load onto non-denaturing PAGE
Resolve complexes & free probe
protein concentration
-
shifted probe
free probe
+
0
EMSA
Resolution of multiple protein/DNA
complexes: Proteins sharing a DNA
binding element
G
A G
A
0 0
B
Deoxyribonuclease I
transcription
factor
5'
3'
3'
5'
*
Deoxyribonuclease I
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
5'
3'
3'
G
A G
0 0
DNAase I Footprinting
Deoxyribonuclease I
transcription
factor
5'
3'
3'
5'
Deoxyribonuclease I
5'
3'
5'
5'
5'
5'
3'
100-300 bp DNA fragment
3'
with unique end-label3' 3'
5'
3'
5'
5'
5'
3'
Incubate
with test protein(s)
3'
Digest3' with DNase I
5'
5'
3'
Run3' digested DNA on PAGE
5'
3'
5'
5'
3'
3'
Summary
Methods to analyze transcription
- detection of mRNAs
- determination of transcriptional start sites
-genome wide expression analysis by microarrays
-reporter assay systems
Methods to determine core promoter and/or enhancer
elements
-promoter mutagenesis
-sequence alignment
Methods to study DNA-protein interactions
-EMSA,
-DNase foot printing
- Chromatin IP
Level
Methods
Transcription
Northern blot
In situ hybridization
RNase protection assay
Reverse transcriptase PCR (RT-PCR)
Western blot
ELISA
Translation
DNA-protein
interaction
EMSA,
DNase I footprinting,
Chromatin immunoprecipitation (ChIP) assay
Protein-protein Pull down assay
interaction
Yeast two hybrid assay
Coimmunoprecipition
Fluorescence resonance energy trnsfer (FRET)