CHMI 4226 - W2009 1

Recombinant DNA TechnologyCHMI 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

M MMboII

1 353

1 078

872

603

310

Cells (e.g. fibroblasts)

Proteinase K + EDTA + SDS

DNAse-free RNAse A

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 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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mRNA

intron ORF

Characterization of a promoter

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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

From: BD-Bioscience

Clone promoter fragments here!

Reporter gene

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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

-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