CH391L/S12/In vitro Selection: Difference between revisions

Revision as of 18:03, 28 January 2012

Overview of in vitro selection

Library Generation

Randomized Oligodeoxynucleotides

Mutagenic PCR

Gene Shuffling

Neutral Drift

Increased Representation

Once diversity is created, the selection must must allow function variants to become a larger percentage of the pool. This step is often the most difficult to design.

Affinity

The easiest function to enrich for is affinity for a ligand. To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand. The best binders affix to the ligand while weaker binders are washed away. Those that remain can be amplified for further round of selection. For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule. For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein. Some examples of this linking are phage display, cell-surface display, and ribosome display.

Protection

Selective Amplification

FACS

Confinement of Function

Binding

Cis-action

Cellularization

===In vitro Compartmentalization=======History

Nucleic Acids

Aptamers

Ellington 1990

Ribozymes

Bartel 1993

Proteins

Antibodies

Accepting suggestions

DNA Modifying Proteins

Tawfik 1998

Other Enzymatic Functions

Griffiths 2003

Self-Replication

Ghadessy 2001

@@ Line 11: / Line 11: @@
 Once diversity is created, the selection must must allow function variants to become a larger percentage of the pool.  This step is often the most difficult to design.
 =====Affinity=====
-The easiest function to enrich for is affinity for a ligand.  To select for binders (aptamers for nucleic acids, antibodies and others for proteins)one exposes the pool of potential binders to a fixed ligand.  The best binders affix to the ligand while weaker binders are washed away.  Those that remain can be amplified for further round of selection.  For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule.  For protein binders, the scheme must include some sort of linking of the information-carrying nucleic acid and the function-carrying protein.  Come examples of this linking are phage-display, cell-surface-display, and ribosome-display.
+The easiest function to enrich for is affinity for a ligand.  To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand.  The best binders affix to the ligand while weaker binders are washed away.  Those that remain can be amplified for further round of selection.  For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule.  For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein.  Some examples of this linking are phage display, cell-surface display, and ribosome display.
 =====Protection=====
 =====Selective Amplification=====