Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique.
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.
Acevedo
Juan Pablo
JP
Centre for Biochemical Engineering and Biotechnology (CIBYB), Department of Chemical Engineering and Biotechnology, Millennium Institute for Cell Dynamics and Biotechnology (ICDB), University of Chile, Beauchef 861, Santiago, Chile. juaceved@ing.uchile.cl
Reyes
Fernando
F
Parra
Loreto P
LP
Salazar
Oriana
O
Andrews
Barbara A
BA
Asenjo
Juan A
JA
D000864:Antarctic Regions; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D016386:Chromosome Walking / Q000379:methods*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004262:DNA Restriction Enzymes; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D006868:Hydrolysis; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D020539:Sequence Analysis, Protein; D012995:Solubility; D020860:Subtilisin / Q000235:genetics; D014871:Water Microbiology
D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D020860:Subtilisin