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Site‐directed combinatorial construction of chimaeric genes: General method for optimizing assembly of gene fragments
Author(s) -
Saftalov Liz,
Smith Peter A.,
Friedman Alan M.,
BaileyKellogg Chris
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20984
Subject(s) - gene , computational biology , computer science , biology , ligation , simulated annealing , genetics , algorithm , microbiology and biotechnology
Site‐directed construction of chimaeric genes by in vitro recombination “mixes‐and‐matches” precise building blocks from multiple parent proteins, generating libraries of hybrids to be tested for structure–function relationships and/or screened for favorable properties and novel enzymatic activities. A direct annealing and ligation method can construct chimaeric genes without requiring sequence identity between parents, except for the short (≈3 nt) sequences of the fragment overhangs used for specific ligation. Careful planning of the assembly process is necessary, though, in order to ensure effective construction of desired fragment assemblies and to avoid undesired assemblies (e.g., repetition of fragments, fragments out of order). We develop algorithms for specific planned ligation of short overhangs (SPLISO) that efficiently explore possible assembly plans, varying the fragment overhangs and the order of ligation steps in the assembly pathway. While there is a combinatorial explosion in the number of possible assembly plans as the number of breakpoints and parent genes increases, we employ a dynamic programming approach to find globally optimal ones in low‐order polynomial time (in practice, taking only seconds for basic assembly plans). We demonstrate the effectiveness of our algorithms in planning the assembly of hybrid libraries, under a variety of experimental options and restrictions, including flexibility in the position and amino acid sequence of breakpoints. Our method promises to enable more effective application of site‐directed recombination to protein investigation and engineering. Proteins 2006. © 2006 Wiley‐Liss, Inc.