Computational and experimental analysis of DNA shuffling.
We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless "junk" DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions.
Maheshri
Narendra
N
Department of Chemical Engineering and Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720-1462, USA.
Schaffer
David V
DV
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D032621:DNA Shuffling / Q000706:statistics & numerical data; D021901:DNA, Intergenic / Q000235:genetics; D049308:Dioxygenases; D019020:Directed Molecular Evolution; D049452:Green Fluorescent Proteins; D007506:Iron-Sulfur Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization; D010105:Oxygenases / Q000235:genetics; D016133:Polymerase Chain Reaction; D013381:Subtilisins / Q000235:genetics; D013816:Thermodynamics
D017931:DNA Primers; D021901:DNA, Intergenic; D007506:Iron-Sulfur Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D010105:Oxygenases; D049308:Dioxygenases; C071575:chlorobenzene dioxygenase; C023172:toluene dioxygenase; C074161:biphenyl-2,3-dioxygenase; D013381:Subtilisins