-The FIND® Library in details
Step 0. Selection of clones to be recombined
The starting material is e.g. gene variants generated by random mutagenesis. The best clones that show the feature looked for, such as improved binding, stability or activity, are selected. Up to ten clones are normally used in a FIND recombination. Two genes are showed in the figure to the right.
Step 1. Preparation of plus (+) and minus (-) ssDNA
ssDNA is produced using for example
- biotinylated gene specific primers and subsequent separation using StreptAvidin columns or
- PCR amplification, with gene specific primers followed by DNA lambda exonuclease digestion.
Step 2. Fragmentation by exonucleases
Pools of the + and - strands are treated with different exonuclease cocktails, respectively, to produce the ssDNA fragments.
Step 3. FIND recombination
The fragmented + and – strands are mixed and polymerase is added. Optionally it is possible to use only the - strand from one variant and the + strands from another (this gives a library guarantied without input background, i.e. no wild type or starting material).
After 25 cycles of hybridization, elongation and denaturation (PCR reaction without adding primers) to create full length dsDNA representing recombined genes, gene specific primers are added (hybridising in the ends amplifying only full length genes) to perform a PCR amplification step to get enough DNA for cloning.
Step 4. Cloning and expression
The libraries are made by cloning into a suitable vector (project specific). Library size (individual clones per microgram of input DNA), recombination frequency and background frequencies are calculated by sequence analysis of randomly selected clones.
Alligator Bioscience has the experience in producing very large protein variant libraries in many different hosts.
- Bacteria (E.coli)
- Incect cells (Drosophilia)
- Mammalian cells (3T3 and COS)