Things to be wary of when using DRIVeR:
DRIVeR uses a generic Poisson model of crossover probabilities and
positions. There are a few caveats that you should be aware of:
Please refer to Patrick W. M., Firth A. E., Blackburn J.M., 2003,
User-friendly algorithms for estimating completeness and diversity in
randomized protein-encoding libraries, Protein Eng., 16,
451-457 for further discussion of DRIVeR.
- Remember to use GLUE, not DRIVeR, when all daughter sequences
are equally likely (e.g. synthetic shuffling and SISDC).
- In the current implementation of DRIVeR, you can not have more
than two different parent sequences.
- As with PEDEL, there is the potential for amplification bias
(see PEDEL caveats here.)
- The two parent sequences are assumed to be highly homologous.
For parent sequences that are homologous at the amino acid level but
divergent at the nucleotide level, crossovers preferentially occur
in regions with greater nucleotide sequence similarity. This bias is
not reflected in the DRIVeR model which, nevertheless, provides a
useful upper bound on library diversity. It has been suggested
(Moore G.L., Maranas C.D., 2002, Nuc. Acids. Res., 30,
2407) that one way to reduce this bias is to synthesize new parent
sequences that maintain the amino acid sequences of the original
parents, but have greater similarity at the level of nucleotides.
In the case of shuffling two epPCR-generated clones, large-scale
sequence dissimilarity will not be an issue.
- Any biases in library construction will decrease the actual
number of distinct variants represented in the library. In such
cases, DRIVeR provides the user with a useful upper bound on the
diversity present in the library.
A good review of the sources of bias in recombination and other
directed evolution protocols can be found in Neylon C., 2004, Chemical
and biochemical strategies for the randomization of protein encoding
DNA sequences: library construction methods for directed evolution,
Nucleic Acids Res., 32, 1448-1459.
Return to DRIVeR server page.
Return to DRIVeR software download page.