Nucleic Acids Research Advance Access originally published online on July 13, 2009
Nucleic Acids Research 2009 37(17):e118; doi:10.1093/nar/gkp561
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Nucleic Acids Research, 2009, Vol. 37, No. 17 e118
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Integrase-directed recovery of functional genes from genomic libraries
1Division of Clinical Integrative Biology, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, S1-26A, Toronto, Ontario M4N 3N5 and 2Department of Laboratory Medicine & Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Canada
*To whom correspondence should be addressed. Tel: +1 416 4806100; Fax: +1 416 4805737; Email: dean.rowe-magnus{at}sri.utoronto.ca
Received March 9, 2009. Revised June 12, 2009. Accepted June 16, 2009.
Large population sizes, rapid growth and 3.8 billion years of evolution firmly establish microorganisms as a major source of the planet's biological and genetic diversity. However, up to 99% of the microorganisms in a given environment cannot be cultured. Culture-independent methods that directly access the genetic potential of an environmental sample can unveil new proteins with diverse functions, but the sequencing of random DNA can generate enormous amounts of extraneous data. Integrons are recombination systems that accumulate open reading frames (gene cassettes), many of which code for functional proteins with enormous adaptive potential. Some integrons harbor hundreds of gene cassettes and evidence suggests that the gene cassette pool may be limitless in size. Accessing this genetic pool has been hampered since sequence-based techniques, such as hybridization or PCR, often recover only partial genes or a small subset of those present in the sample. Here, a three-plasmid genetic strategy for the sequence-independent recovery of gene cassettes from genomic libraries is described and its use by retrieving functional gene cassettes from the chromosomal integron of Vibrio vulnificus ATCC 27562 is demonstrated. By manipulating the natural activity of integrons, we can gain access to the caches of functional genes amassed by these structures.