Methods for in vitro joining and combinatorial assembly of nucleic acid molecules
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United States of America Patent
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Mar 3, 2015
Grant Date -
Feb 11, 2010
app pub date -
Feb 13, 2009
filing date -
Feb 15, 2008
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Abstract
The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.
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