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Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique

Paper ID Volume ID Publish Year Pages File Format Full-Text
24802 43538 2008 10 PDF Available
Title
Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique
Abstract

The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3′ adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3′ overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria.

Keywords
Genome walking; Chromosome walking; CODEHOP; Antarctic; Bacteria; Protease; Subtilisin
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Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 133, Issue 3, 1 February 2008, Pages 277–286
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us