Related Experiment Videos
Predicting out-of-sequence reassembly in DNA shuffling
Gregory L Moore1, Costas D Maranas
1Department of Chemical Engineering, The Pennsylvania State University, 112 Fenske Laboratory, University Park, PA, 16802, U.S.A.
Journal of Theoretical Biology
|October 24, 2002
Summary
This study analyzes out-of-sequence reassembly in DNA shuffling, finding it generates non-functional proteins. Decreasing fragment length increases these events, with a minimum near 55°C.
Area of Science:
- Molecular Biology
- Biotechnology
- Bioinformatics
Background:
- DNA shuffling is a powerful gene evolution technique.
- Out-of-sequence reassembly in DNA shuffling can lead to non-functional proteins with undesirable missing or repetitive regions.
Purpose of the Study:
- To develop an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments.
- To understand the factors influencing out-of-sequence annealing events.
Main Methods:
- Utilized the e Shuffle framework, incorporating equilibrium thermodynamics and complete sequence information.
- Modeled the DNA reassembly process computationally.
- Conducted an in silico case study using subtilase sequences.
Main Results:
- Significant sequence identity between parental DNA fragments increases out-of-sequence annealing.
- Shorter DNA fragment lengths correlate with a higher frequency of these undesirable events.
- Out-of-sequence annealing events were minimized near an annealing temperature of 55°C.
Conclusions:
- The developed analysis provides a method to predict and understand out-of-sequence reassembly in DNA shuffling.
- Fragment length and annealing temperature are key factors influencing the occurrence of these events.
- Further optimization of DNA shuffling protocols can minimize the generation of non-functional protein variants.