| | |
Summary: Analysis of Shuffled Gene Libraries
John M. Joern, Peter Meinhold and Frances H. Arnold*
Division of Chemistry and
Chemical Engineering 210-41
California Institute of
Technology, Pasadena
CA 91125, USA
In vitro recombination of homologous genes (family shuf¯ing) has been
proposed as an effective search strategy for laboratory evolution of genes
and proteins. Few data are available, however, on the composition of
shuf¯ed gene libraries, from which one could assess the ef®ciency of
recombination and optimize protocols. Here, probe hybridization is used
in a macroarray format to analyze chimeric DNA libraries created by
DNA shuf¯ing. Characterization of hundreds of shuf¯ed genes encoding
dioxygenases has elucidated important biases in the shuf¯ing reaction.
As expected, crossovers are favored in regions of high sequence identity.
A sequence-based model of homologous recombination that captures this
observed bias was formulated using the experimental results. The chi-
meric genes were found to show biases in the incorporation of sequences
from certain parents, even before selection. Statistically different patterns
|
