A cDNA library represents a sample of the mRNA purified from a particular source (either a collection of cells, a particular tissue, or an entire organism), which has been converted back to a DNA template by the use of the enzyme reverse transcriptase.
It thus represents the genes that were being actively transcribed in that particular source under the physiological, developmental, or environmental conditions that existed when the mRNA was purified.
[4] Alternatively, mutations can be targeted to specific codons during de novo synthesis or saturation mutagenesis to construct one or more point mutants of a gene in a controlled way.
The expressed proteins from these libraries can then be screened for variants which exhibit favorable properties (e.g. stability, binding affinity or enzyme activity).
This can be repeated in cycles of creating gene variants and screening the expression products in a directed evolution process.
However, the high transfection efficiency achieved by using viruses (often phages) makes them useful for packaging the vector (with the ligated insert) and then introducing them into the bacterial (or yeast) cell.
Additionally, for cDNA libraries, a system using the Lambda Zap II phage, ExAssist, and 2 E. coli species has been developed.
Site saturation mutagenesis
is a type of
site-directed mutagenesis
. This image shows the saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants of the isoleucine at position 5 are shown below.
How DNA libraries generated by
random mutagenesis
sample sequence space. The amino acid substituted into a given position is shown. Each dot or set of connected dots is one member of the library. Error-prone PCR randomly mutates some residues to other amino acids. Alanine scanning replaces each residue of the protein with alanine, one-by-one. Site saturation substitutes each of the 20 possible amino acids (or some subset of them) at a single position, one-by-one.
Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red). Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below).
Workflow for screening a synthetic library to identify cells producing a chemical of interest.
