Transcript How do we determine a genes function?

So You Found
a Gene ?
NOW WHAT ??
How do we determine a genes function?
We can infer certain roles based on
sequence comparisons (Couple Weeks Ago)
These roles are not concrete without
experimental data
For Example:the NEW protein is a kinase
(based on sequence) but without showing
that the kinase domain is necessary for
function this is not confirmed.
How would this be possible using the
techniques we have available?
How do we determine a genes function?
The function of a gene (or Section of
a Gene) can be determined using Two
methods along with Transgenesis.
Site-directed Mutagenesis:
Single/Double Amino Acid Changes
Small Deletions
Domain Mutagenesis:
Large sections (Domains) of a gene
product are removed
Site-Directed Mutagenesis
Involves Specific Base-pair changes
in a DNA sequence.
Can be done in several ways:
PCR mediated
Vector mediated
Very useful in determining the role
of active site residues, residues that
are believed to be phosphorylated,
etc.
How does it work ?
In site-directed mutagenesis Primers are deisgned
which contain a small area that is different and a
large area that is identical to the ‘Normal’Gene
Sequence.
When a Polymerase incorporates this primer the
change is made in one copy of the resulting
products.
Some methods then select for the ‘Altered’
Version.
T
S
R
A C G T C T A G A
A C G C C T A G A
T
A
R
Vector Mediated
Removing Domains
NEW Kinase
Kinase
A
C
Design PCR Primers which ‘JUMP’ B
Protein
without Kinase
Domain
A
C