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Recombinant DNA Technology
CHMI 4226
Week of March 16, 2009
Isolating and characterizing genes
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Genes structure
• Promoter:
– DNA sequence located
upstream of the gene;
– Bind transcription factors
and RNA polymerase;
– indicates where
transcription should
begin (TSS: transcription
start site).
• Intron splicing
sequences:
– Introns always (well,
almost…) begin with GT
and end with AG.
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Why clone genes
• Identifying and characterizing promoter
sequences which participate in regulating
transcription;
• Identification and characterization of the
introns and exons (size, number,
involvement in regulation of gene
expression).
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Genomic library
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Genomic DNA
MboII
M
Cells (e.g.
fibroblasts)
Proteinase K +
EDTA + SDS
M
1 353
1 078
872
DNAse-free
RNAse A
603
310
Extraction
1. phenol
2. chloroform
Ethanol precipitation
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Genomic library – cosmid vectors
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Walking on the chromosome
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Example of gene isolation:
Mouse Heme oxygenase
J. Biol. Chem. 1994. 269[2]: 1001-1009
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mRNA
intron
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ORF
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Example of gene isolation:
Mouse Heme oxygenase
J. Biol. Chem. 1994. 269[2]: 1001-1009
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Example of gene isolation:
Identifying intron-exon boundaries
S1 nuclease mapping
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Using S1 nuclease mapping to locate
the transcription start site
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Mapping the transcription start site
using “primer extension”
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Example of gene isolation:
Mouse Heme oxygenase
J. Biol. Chem. 1994. 269[2]: 1001-1009
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Characterization of a promoter
mRNA
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intron
ORF
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Promoter bashing
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Linker Scanning Mutagenesis
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Characterization of a promoter
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Characterization of a promoter
using reporter genes
Clone promoter fragments here!
Reporter gene
From: BD-Bioscience
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Characterization of a promoter
using reporter genes
•
Chemiluminescence:
– Generation of light through an enzymatic reaction– « artificial system»
– E.g. B-galactosidase, horseradish peroxidase
•
Bioluminescence:
– Generation of light through an enzymatic reaction– « natural system»
– E.g. luciferase
•
Autofluorescence:
– E.g. Green fluorescent protein
•
Fluorescence
•
Radioactivity
•
Generation of a chromophore (colored compound)
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Reporter
gene
In vitro assay In vivo
assay
Strength
Weaknesses
Limit of
detection
(molecules)
CAT
1.Chromatography.
2.Fluorescence
3. ELISA
None
-Stable
- minimal endogenous activity
-Labor intensive
-Radioactivity
-Low sensitivity
-Low Dynamic range (2
OM)
- Short half life of
mRNA
5-10 x 107
Luciferase
Bioluminescence
Bioluminescence
-Non radioactive
-Large dynamic range (4
orders of magnitude)
- minimal endogenous activity
- Short half life of
protein
- Expensive
instrumentation
- Low reproducibility
1-2,5 x 105
b-gal
1.Colorimetric
2. Fluorescence
3. Chemilumin.
1. Histochemistry
2. Biolumin.
-Non radioactive
-Large dynamic range (5-6
OM)
- High endogenous
activity
- Expensive
instrumentation
104 -105
(chemilumin.)
SEAP
1. Colorimetric
2. Bioluminescence.
3. Chemiluminescence
None
-Non radioactive
-Large dynamic range (4 OM)
- High endogenous
activity
- dependent on the
secretory activity of the
cell line used
104 -105
(chemilumin.)
GFP
Fluorescence
Fluorescence
-Works in living cells
- non-toxic
-No photobleaching
-Signal too faint for
some applications
n/a
Source: Current Protocols in Molecular Biology. Table 9.6.1
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Characterization of a promoter
using reporter genes
http://www.promega.com/pnotes/70/7618_25/7618_25.html
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Example of gene isolation:
Mouse Heme oxygenase
J. Biol. Chem. 1994. 269[2]: 1001-1009
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Characterization of a promoter
using reporter genes
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Characterization of a promoter
using reporter genes
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Example of gene isolation:
Mouse Heme oxygenase
J. Biol. Chem. 1994. 269[2]: 1001-1009
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