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 Primer...
Published in: | Journal of Biotechnology |
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Format: | Article in Journal/Newspaper |
Language: | English |
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Online Access: | http://hdl.handle.net/10533/196745 https://doi.org/10.1016/j.jbiotec.2007.10.004 |
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ftconicyt:oai:localhost:10533/196745 2023-05-15T13:53:09+02:00 Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique ASENJO-DE LEUZE, JUAN SALAZAR-AGUIRRE, ORIANA ACEVEDO-COX, JUAN REYES-B., FERNANDO ANDREWS-FARROW, BARBARA PARRA-ATALA, LORETO NLD AMSTERDAM http://hdl.handle.net/10533/196745 https://doi.org/10.1016/j.jbiotec.2007.10.004 ENG eng ELSEVIER SCIENCE BV instname: Conicyt reponame: Repositorio Digital RI2.0 info:eu-repo/grantAgreement/Fondef/D04I1374 info:eu-repo/semantics/dataset/hdl.handle.net/10533/93477 http://hdl.handle.net/10533/196745 doi:10.1016/j.jbiotec.2007.10.004 wos: WOS:000252632200002 eissn: 0 issn: 0168-1656 info:eu-repo/semantics/openAccess JOURNAL OF BIOTECHNOLOGY info:eu-repo/semantics/article ftconicyt https://doi.org/10.1016/j.jbiotec.2007.10.004 2019-09-07T08:18:01Z 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. (C) 2007 Elsevier B.V. All rights reserved. Article in Journal/Newspaper Antarc* Antarctic Repositorio Digital Conicyt RI 2.0 (Comisión Nacional de Investigación Científica y Tecnológica) Antarctic Journal of Biotechnology 133 3 277 286 |
institution |
Open Polar |
collection |
Repositorio Digital Conicyt RI 2.0 (Comisión Nacional de Investigación Científica y Tecnológica) |
op_collection_id |
ftconicyt |
language |
English |
description |
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. (C) 2007 Elsevier B.V. All rights reserved. |
format |
Article in Journal/Newspaper |
author |
ASENJO-DE LEUZE, JUAN SALAZAR-AGUIRRE, ORIANA ACEVEDO-COX, JUAN REYES-B., FERNANDO ANDREWS-FARROW, BARBARA PARRA-ATALA, LORETO |
spellingShingle |
ASENJO-DE LEUZE, JUAN SALAZAR-AGUIRRE, ORIANA ACEVEDO-COX, JUAN REYES-B., FERNANDO ANDREWS-FARROW, BARBARA PARRA-ATALA, LORETO Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
author_facet |
ASENJO-DE LEUZE, JUAN SALAZAR-AGUIRRE, ORIANA ACEVEDO-COX, JUAN REYES-B., FERNANDO ANDREWS-FARROW, BARBARA PARRA-ATALA, LORETO |
author_sort |
ASENJO-DE LEUZE, JUAN |
title |
Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
title_short |
Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
title_full |
Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
title_fullStr |
Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
title_full_unstemmed |
Cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
title_sort |
cloning of complete genes for novel hydrolytic enzymes from antarctic sea water bacteria by use of an improved genome walking technique |
publisher |
ELSEVIER SCIENCE BV |
url |
http://hdl.handle.net/10533/196745 https://doi.org/10.1016/j.jbiotec.2007.10.004 |
op_coverage |
NLD AMSTERDAM |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
JOURNAL OF BIOTECHNOLOGY |
op_relation |
instname: Conicyt reponame: Repositorio Digital RI2.0 info:eu-repo/grantAgreement/Fondef/D04I1374 info:eu-repo/semantics/dataset/hdl.handle.net/10533/93477 http://hdl.handle.net/10533/196745 doi:10.1016/j.jbiotec.2007.10.004 wos: WOS:000252632200002 eissn: 0 issn: 0168-1656 |
op_rights |
info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.1016/j.jbiotec.2007.10.004 |
container_title |
Journal of Biotechnology |
container_volume |
133 |
container_issue |
3 |
container_start_page |
277 |
op_container_end_page |
286 |
_version_ |
1766258129935269888 |