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Efficient recovery of environmental DNA for expression cloning by indirect extraction methods
Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability...
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| Published in: | FEMS microbiology ecology 2003-05, Vol.44 (2), p.153-163 |
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| container_end_page | 163 |
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| container_title | FEMS microbiology ecology |
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| creator | Gabor, Esther M de Vries, Erik J Janssen, Dick B |
| description | Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability for expression cloning in Escherichia coli. Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used ( |
| doi_str_mv | 10.1016/S0168-64960200462-2 |
| format | article |
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Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used (<8% of eukaryotic DNA by indirect methods versus 61–93% by direct lysis protocols). Considering the higher purity, i.e. higher cloning efficiency of DNA isolated by indirect methods, similar numbers of clones carrying prokaryotic inserts could be produced by either strategy. Gene banks prepared from directly extracted DNA, however, are expected to contain large portions of clones with eukaryotic inserts, whereas those constructed from indirectly isolated DNA should mainly contain inserts of bacterial origin. As eukaryotic genetic information is generally not expressed in bacterial host organisms but increases the library size, our findings suggest that the use of indirect DNA isolation methods allows the construction of environmental gene banks of superior quality.</description><identifier>ISSN: 0168-6496</identifier><identifier>EISSN: 1574-6941</identifier><identifier>DOI: 10.1016/S0168-64960200462-2</identifier><language>eng</language><publisher>Delft: Oxford University Press</publisher><subject>Activated sludge ; Bacteria ; Cloning ; Construction methods ; Deoxyribonucleic acid ; DNA ; E coli ; Ecology ; Electrophoresis ; Environmental DNA ; Gel electrophoresis ; Gene banks ; Gene expression ; Inserts ; Loam soils ; Lysis ; Microbiology ; Soils</subject><ispartof>FEMS microbiology ecology, 2003-05, Vol.44 (2), p.153-163</ispartof><rights>2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. 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Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used (<8% of eukaryotic DNA by indirect methods versus 61–93% by direct lysis protocols). Considering the higher purity, i.e. higher cloning efficiency of DNA isolated by indirect methods, similar numbers of clones carrying prokaryotic inserts could be produced by either strategy. Gene banks prepared from directly extracted DNA, however, are expected to contain large portions of clones with eukaryotic inserts, whereas those constructed from indirectly isolated DNA should mainly contain inserts of bacterial origin. As eukaryotic genetic information is generally not expressed in bacterial host organisms but increases the library size, our findings suggest that the use of indirect DNA isolation methods allows the construction of environmental gene banks of superior quality.</description><subject>Activated sludge</subject><subject>Bacteria</subject><subject>Cloning</subject><subject>Construction methods</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>E coli</subject><subject>Ecology</subject><subject>Electrophoresis</subject><subject>Environmental DNA</subject><subject>Gel electrophoresis</subject><subject>Gene banks</subject><subject>Gene expression</subject><subject>Inserts</subject><subject>Loam soils</subject><subject>Lysis</subject><subject>Microbiology</subject><subject>Soils</subject><issn>0168-6496</issn><issn>1574-6941</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><recordid>eNqNi8FOAjEURRujiQPyBW5ewnrgtdOpsCSKceUGtoQMwyuWDH3YFiJ_b00Iazf3JvecK8SzxJFEacaLHJPS6KlBhaiNKtWdKGT9oksz1fJeFDfhUfRi3CPKutJYiNXcWtc68gkCtXymcAG2QP7sAvtD3psO3j5nYDkA_RwDxejYQ9uxd34Hmws4v3X5mzJOoWnTHz5Q-uJtfBIPtukiDa7dF8P3-fL1ozwG_j5RTOs9n4LPaK0qWSulJ6ir_1m_ueRLWQ</recordid><startdate>20030501</startdate><enddate>20030501</enddate><creator>Gabor, Esther M</creator><creator>de Vries, Erik J</creator><creator>Janssen, Dick B</creator><general>Oxford University Press</general><scope>3V.</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope></search><sort><creationdate>20030501</creationdate><title>Efficient recovery of environmental DNA for expression cloning by indirect extraction methods</title><author>Gabor, Esther M ; 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Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used (<8% of eukaryotic DNA by indirect methods versus 61–93% by direct lysis protocols). Considering the higher purity, i.e. higher cloning efficiency of DNA isolated by indirect methods, similar numbers of clones carrying prokaryotic inserts could be produced by either strategy. Gene banks prepared from directly extracted DNA, however, are expected to contain large portions of clones with eukaryotic inserts, whereas those constructed from indirectly isolated DNA should mainly contain inserts of bacterial origin. As eukaryotic genetic information is generally not expressed in bacterial host organisms but increases the library size, our findings suggest that the use of indirect DNA isolation methods allows the construction of environmental gene banks of superior quality.</abstract><cop>Delft</cop><pub>Oxford University Press</pub><doi>10.1016/S0168-64960200462-2</doi></addata></record> |
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| identifier | ISSN: 0168-6496 |
| ispartof | FEMS microbiology ecology, 2003-05, Vol.44 (2), p.153-163 |
| issn | 0168-6496 1574-6941 |
| language | eng |
| recordid | cdi_proquest_journals_2315224804 |
| source | Oxford Open Access Journals |
| subjects | Activated sludge Bacteria Cloning Construction methods Deoxyribonucleic acid DNA E coli Ecology Electrophoresis Environmental DNA Gel electrophoresis Gene banks Gene expression Inserts Loam soils Lysis Microbiology Soils |
| title | Efficient recovery of environmental DNA for expression cloning by indirect extraction methods |
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