Topic I - Gene Function Research Technique

Reporter Gene

A gene whose expression is easy to detect

Examples: Luciferase (light), GFP (fluorescence), Beta-galactosidase (colour)

-Sensitive; Easy to detect even at low levels of expression

Reporter Gene Assay

Promoter sequence of gene of interest used

-Gene of interest is replaced with a reporter gene, the promoter then transcribes the reporter gene, activity can then be detected (reporter gene mRNA → protein = level of activity)

Promoter Deletion Analysis

Objective: Identify location of potential regulatory elements for a gene of interest

Description: Promoter and upstream DNA are fused to a reporter gene. Series of deletion fragments used to observe whether a change in expression of the reporter gene is observed

-Molecular cloning is used to create promoter fusions

-Introduced into cells, expression is then measured

No gene expression = Core promoter not present in the fragment

Electrophoretic Mobility Shift Assay (EMSA)

In vitro technique

Objective: Determine if a protein can bind to a specific DNA fragment

Example; protein being a transcription factor, DNA fragment is a promoter region

Description: Incubate a short, labelled DNA fragment with a protein of interest, run them on a gel to see if the protein binds

-Binding causes a mobility shift

Steps of EMSA

1. Generate many labelled copies of DNA fragments using PCR (ex; radioactive primer to generate labelled copies)

2. Incubate DNA with transcription factor of interest

3. Run on a gel to determine if there is a mobility shift

-Molecule weight of DNA molecule increases if the protein can bind

-Should be higher on the gel than the control (non-bound DNA fragment)

-Control is DNA only, confirms mobility shift occurs only when protein is present

DNase Footprinting

Objective: Determines the precise DNA sequence that is bound by a transcription factor for a gene of interest

-in-vitro technique

Example: Identifying sequence of the regulatory element + location of regulatory element

Description: DNA fragment is labelled via radioactive primers in PCR

-Sequence of DNA upstream of a gene is incubated with the transcription factor

-After the transcription factor has bound, DNase I is used to cut unprotected (no protein) DNA to determine where transcription factor is bound

Steps of DNase Footprinting

1. Use DNase I - Endonuclease cuts free DNA (not bound by protein)

• DNase makes a single cut in DNA molecule, one end of the molecule contains the radioactive label and the other doesn’t (won’t be detected)

2. Different sized fragments produced, visualized via gel electrophoresis

• Control: cut DNA that is not bound to protein

• Fragments of DNA will be missing, tells us the sequence that the protein bound to (footprint)

• Can pair with sanger sequencing to determine the exact sequence of the fragment

Chromatin Immunoprecipitation (ChIP)

Objective: Determine where a transcription factor (or other DNA-binding protein) of interest binds in vivo

-Used to identify all DNA binding sites (regulatory elements) for a particular transcription factor in the genome

Description: Isolate chromatin (DNA + all bound protein)

-Shear into smaller fragments

-Use antibodies that bind to a transcription factor (or protein of interest) to isolate proteins and bound DNA

Steps of ChIP

1. Covalently crosslink DNA and proteins together (glues proteins onto DNA)

2. Isolate chromatin from cells

3. Fragment chromatin

4. Purify with antiboody that binds to protein/transcription factor of interest

5. Isolate DNA

6. Identify the DNA

-Next-generation sequencing (all genes regulated by transcription factor)

-PCR with gene specific primers