Predicting out-of-sequence reassembly in DNA shuffling.
We present an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments. Out-of-sequence annealing events are undesirable since they typically encode non-functional proteins with missing or repetitive regions. The approach builds on the e Shuffle framework for the prediction of crossover formation using equilibrium thermodynamics and complete sequence information to model the reassembly process. An in silico case study of a set of subtilases reveals that, as expected, the presence of significant sequence identity between distant portions of the parental sequences gives rise to out-of-sequence annealing events that upon reassembly generate sequences with missing or repetitive DNA segments. The frequency of these events increases as the fragment length decreases. Interestingly, out-of-sequence annealing events are at a minimum near the annealing temperature of 55 degrees C used in the original DNA shuffling protocol. Neither parental sequence identity nor number of shuffled parents significantly alter the extent of out-of-sequence reassembly
Moore
Gregory L
GL
Department of Chemical Engineering, The Pennsylvania State University, 112 Fenske Laboratory, University Park, PA, 16802, U.S.A.
Maranas
Costas D
CD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D032621:DNA Shuffling; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009695:Nucleic Acid Renaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D013696:Temperature