CH391L/S12/In vitro Selection

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(Mutagenic PCR)
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=====Mutagenic PCR=====
=====Mutagenic PCR=====
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A wild-type starting sequence can be concomitantly mutated and amplified using mutagenic PCR.  Infidelity can be achieved using an error-prone DNA polymerase, increasing the magnesium content, including manganese, using non-canonical dNTPs (e.g. 8-oxo-GTP), or altering the ratio of the dNTPs.
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=====Gene Shuffling=====
=====Gene Shuffling=====
=====Neutral Drift=====
=====Neutral Drift=====

Revision as of 13:43, 29 January 2012

Contents

Overview of in vitro selection

Library Generation

Randomized Oligodeoxynucleotides

Randomized oligodeoxynucleotides are generated using traditional solid-phase phosphoramidite chemistry. At the "N" positions, an equimolar mixture of the 4 bases are used, thus giving an equal probability of each base at that position on a given nascent oligodeoxynucleotide.

If there is a known, functional sequence that can serve as a starting point, then a pool can be mildly randomized (yielding a "doped" pool). In a doped pool, a mixture of all four bases are used, but the molarity is skewed to favor the wild-type base at each given position.

Mutagenic PCR

A wild-type starting sequence can be concomitantly mutated and amplified using mutagenic PCR. Infidelity can be achieved using an error-prone DNA polymerase, increasing the magnesium content, including manganese, using non-canonical dNTPs (e.g. 8-oxo-GTP), or altering the ratio of the dNTPs.

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. In Ellington and Szostak (1990), A DNA library was created such a T7 promoter drives expression of an N100 RNA pool, flanked by constant regions. The pool is passed through a column with a bound ligand. Species that bind the ligand are retained on the column and weaker binders are washed away. Binders are eluted from the column and amplified to begin a new round.

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.

Selective Amplification

Bartel 1993

Protection

The first in vitro evolved protein functions involved modification of the nucleic acid species that encoded it. Perhaps the first such function was protection of the DNA template. In Tawfik and Griffiths (1998) The template encodes HaeIII methyl transferase. Upon transcription and translation in bacterial lysate, active methyltransferase methylate HaeIII recognition sequences in the gene. The methylated genes are then protected from digestion by HaeIII endonuclease. Undigested templates are then amplified before the next round. The key to this experiment is the use of in vitro compartmentalization (described below)which allow an active methyltransferase to methylate its parent template, but not other templates in the pool.

FACS

Griffiths 2003 Griffiths 2003 Schema

Confinement of Function

Binding
Cis-action
Cellularization
In vitro Compartmentalization

References

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