Controlled Randomization
(up to 1011 variants)
unbiased random substitutions with defined frequency
The problem:
Many approaches adopted to date to identify improved proteins make use of error-prone PCR to create initial diversity. However, it is difficult to control reaction parameters to get close to a preset frequency of substitutions per kilobase. Furthermore, most substitutions generated by Taq polymerase are transitions, making the character of amino acid exchanges profoundly biased.
The solution:
In vitro library synthesis using GENEART technology can introduce random substitutions on a very controlled level. The effective frequency of substitutions can be set very accurately and the type of substitutions can be adjusted to favor transversions as opposed to transitions.
The randomized part of the gene can cover the complete open reading frame or one or more regions thereof.
Deliverables:
Controlled randomization libraries can be delivered in various formats:
- non-amplified library – up to 1011 molecules as verified by real-time PCR.
- amplified library – 5 µg of linear DNA ready to clone via 5' and 3' restriction sites.
- cloned library – 10 µg of plasmid DNA, library subcloned into customer's vector. Includes glycerol stock of total library transformants.
Quality control:
Amplified and cloned library pools will be bulk-sequenced. In addition, a peer group of 48, 96 or 192 individual transformants will be sequence-verified. The statistical analysis of the data obtained includes:
determining the correctness of the non-degenerated part of the sequence
checking for even distribution of nucleotides at the degenerated sites
checking library size via real-time PCR to verify the number of distinct molecules in the non-amplified library
GENEART offers a guaranteed threshold for all three of these critical parameters.
Fill out our online quotation form to get a quote for your specific project.
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