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 dataFor 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 ChangesSmall Deletions

Domain Mutagenesis:Large sections (Domains) of a gene product are removed

Site-Directed MutagenesisInvolves Specific Base-pair changes in a DNA sequence.Can be done in several ways:

PCR mediatedVector 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.

A C G T C T A G A

A C G C C T A G AA

T R

T

S

R

Vector Mediated

Removing DomainsA Kinase CNEW Kinase

Design PCR Primers which ‘JUMP’ B

A CProtein without Kinase Domain