Mutant Library Complexity Tool
When using a randomized mutant library, it is important to ensure that the library actually covers all possible variants. This tool helps to calculate the complexity of a library. Start by choosing the number of randomized amino acid positions:
| Number of randomized amino acids: |
|---|
Desired probability that each variant is represented at least once in the library:
| Synthesis Scale of DNA | 40 nmol of residue per coupling | |
| Minimum amount of DNA end product | 10nmol |
| Conventional library | Entelechon codon-precision library | ||
|---|---|---|---|
| Minimal | Maximal | ||
| Total number of combinations | |||
| Required dsDNA molecules to reach desired probability | |||
| Required amount in mol | |||
| Safety margin (ratio of actual molecules vs. required number): | |||
Explanation
The number of variants (possible permutations of the randomized positions) is determined by how many amino acids are represented at each randomized position. A library should contain enough molecules in order to represent each variant at least once. Given a desired probability that this is the case, the number of required molecules can be calculated.
Since the library is based on a synthesis scale of 40nmol, with an effective yield of 10nmol of end product, it will cover all variants as long as the total number of required molecules is below 10nmol. This tool calculates the ratio of the actually required number of molecules and of the 10nmol yield as the ‘safety margin’. Since there are several ways in which the actual complexity can be reduced (e.g. selection effects, adsorption of molecules to the vessel wall, incomplete turnover into the full-length product), we recommend a safety margin of 1000-fold.
The tool compares conventional libraries – based on single randomized nucleotides – with Entelechon’s codon precision libraries. As conventional libraries cover codons by three independently randomized nucleotides, they usually incorporate additional codon variants, often including stop codons and undesired amino acids. The tool compares the minimal and maximal number of additional variants – which depend on the exact choice of desired amino acids, of course – with the lower number of combinations when using a codon-precision library. This reduction in complexity can save significant amounts of money and effort when processing and screening the library.