Flotation as a tool for indirect DNA extraction from soil
Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Parachin, Nádia Skorupa [verfasserIn] |
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| Format: |
Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2010 |
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| Schlagwörter: |
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| Anmerkung: |
© Springer-Verlag 2010 |
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| Übergeordnetes Werk: |
Enthalten in: Applied microbiology and biotechnology - Springer-Verlag, 1984, 87(2010), 5 vom: 09. Juni, Seite 1927-1933 |
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| Übergeordnetes Werk: |
volume:87 ; year:2010 ; number:5 ; day:09 ; month:06 ; pages:1927-1933 |
| Links: |
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| DOI / URN: |
10.1007/s00253-010-2691-3 |
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OLC2050730063 |
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| 520 | |a Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. | ||
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10.1007/s00253-010-2691-3 doi (DE-627)OLC2050730063 (DE-He213)s00253-010-2691-3-p DE-627 ger DE-627 rakwb eng 570 VZ 12 ssgn BIODIV DE-30 fid Parachin, Nádia Skorupa verfasserin aut Flotation as a tool for indirect DNA extraction from soil 2010 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Springer-Verlag 2010 Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. DNA extraction Soil Humic acid and flotation Schelin, Jenny aut Norling, Börje aut Rådström, Peter aut Gorwa-Grauslund, Marie F. aut Enthalten in Applied microbiology and biotechnology Springer-Verlag, 1984 87(2010), 5 vom: 09. Juni, Seite 1927-1933 (DE-627)129942634 (DE-600)392453-1 (DE-576)015507750 0175-7598 nnns volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 https://doi.org/10.1007/s00253-010-2691-3 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_21 GBV_ILN_23 GBV_ILN_40 GBV_ILN_69 GBV_ILN_70 GBV_ILN_100 GBV_ILN_130 GBV_ILN_147 GBV_ILN_267 GBV_ILN_2004 GBV_ILN_2018 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4307 AR 87 2010 5 09 06 1927-1933 |
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10.1007/s00253-010-2691-3 doi (DE-627)OLC2050730063 (DE-He213)s00253-010-2691-3-p DE-627 ger DE-627 rakwb eng 570 VZ 12 ssgn BIODIV DE-30 fid Parachin, Nádia Skorupa verfasserin aut Flotation as a tool for indirect DNA extraction from soil 2010 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Springer-Verlag 2010 Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. DNA extraction Soil Humic acid and flotation Schelin, Jenny aut Norling, Börje aut Rådström, Peter aut Gorwa-Grauslund, Marie F. aut Enthalten in Applied microbiology and biotechnology Springer-Verlag, 1984 87(2010), 5 vom: 09. Juni, Seite 1927-1933 (DE-627)129942634 (DE-600)392453-1 (DE-576)015507750 0175-7598 nnns volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 https://doi.org/10.1007/s00253-010-2691-3 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_21 GBV_ILN_23 GBV_ILN_40 GBV_ILN_69 GBV_ILN_70 GBV_ILN_100 GBV_ILN_130 GBV_ILN_147 GBV_ILN_267 GBV_ILN_2004 GBV_ILN_2018 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4307 AR 87 2010 5 09 06 1927-1933 |
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10.1007/s00253-010-2691-3 doi (DE-627)OLC2050730063 (DE-He213)s00253-010-2691-3-p DE-627 ger DE-627 rakwb eng 570 VZ 12 ssgn BIODIV DE-30 fid Parachin, Nádia Skorupa verfasserin aut Flotation as a tool for indirect DNA extraction from soil 2010 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Springer-Verlag 2010 Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. DNA extraction Soil Humic acid and flotation Schelin, Jenny aut Norling, Börje aut Rådström, Peter aut Gorwa-Grauslund, Marie F. aut Enthalten in Applied microbiology and biotechnology Springer-Verlag, 1984 87(2010), 5 vom: 09. Juni, Seite 1927-1933 (DE-627)129942634 (DE-600)392453-1 (DE-576)015507750 0175-7598 nnns volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 https://doi.org/10.1007/s00253-010-2691-3 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_21 GBV_ILN_23 GBV_ILN_40 GBV_ILN_69 GBV_ILN_70 GBV_ILN_100 GBV_ILN_130 GBV_ILN_147 GBV_ILN_267 GBV_ILN_2004 GBV_ILN_2018 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4307 AR 87 2010 5 09 06 1927-1933 |
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10.1007/s00253-010-2691-3 doi (DE-627)OLC2050730063 (DE-He213)s00253-010-2691-3-p DE-627 ger DE-627 rakwb eng 570 VZ 12 ssgn BIODIV DE-30 fid Parachin, Nádia Skorupa verfasserin aut Flotation as a tool for indirect DNA extraction from soil 2010 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Springer-Verlag 2010 Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. DNA extraction Soil Humic acid and flotation Schelin, Jenny aut Norling, Börje aut Rådström, Peter aut Gorwa-Grauslund, Marie F. aut Enthalten in Applied microbiology and biotechnology Springer-Verlag, 1984 87(2010), 5 vom: 09. Juni, Seite 1927-1933 (DE-627)129942634 (DE-600)392453-1 (DE-576)015507750 0175-7598 nnns volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 https://doi.org/10.1007/s00253-010-2691-3 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_21 GBV_ILN_23 GBV_ILN_40 GBV_ILN_69 GBV_ILN_70 GBV_ILN_100 GBV_ILN_130 GBV_ILN_147 GBV_ILN_267 GBV_ILN_2004 GBV_ILN_2018 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4307 AR 87 2010 5 09 06 1927-1933 |
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10.1007/s00253-010-2691-3 doi (DE-627)OLC2050730063 (DE-He213)s00253-010-2691-3-p DE-627 ger DE-627 rakwb eng 570 VZ 12 ssgn BIODIV DE-30 fid Parachin, Nádia Skorupa verfasserin aut Flotation as a tool for indirect DNA extraction from soil 2010 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Springer-Verlag 2010 Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. DNA extraction Soil Humic acid and flotation Schelin, Jenny aut Norling, Börje aut Rådström, Peter aut Gorwa-Grauslund, Marie F. aut Enthalten in Applied microbiology and biotechnology Springer-Verlag, 1984 87(2010), 5 vom: 09. Juni, Seite 1927-1933 (DE-627)129942634 (DE-600)392453-1 (DE-576)015507750 0175-7598 nnns volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 https://doi.org/10.1007/s00253-010-2691-3 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_21 GBV_ILN_23 GBV_ILN_40 GBV_ILN_69 GBV_ILN_70 GBV_ILN_100 GBV_ILN_130 GBV_ILN_147 GBV_ILN_267 GBV_ILN_2004 GBV_ILN_2018 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4277 GBV_ILN_4305 GBV_ILN_4307 AR 87 2010 5 09 06 1927-1933 |
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Enthalten in Applied microbiology and biotechnology 87(2010), 5 vom: 09. Juni, Seite 1927-1933 volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 |
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Enthalten in Applied microbiology and biotechnology 87(2010), 5 vom: 09. Juni, Seite 1927-1933 volume:87 year:2010 number:5 day:09 month:06 pages:1927-1933 |
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Parachin, Nádia Skorupa @@aut@@ Schelin, Jenny @@aut@@ Norling, Börje @@aut@@ Rådström, Peter @@aut@@ Gorwa-Grauslund, Marie F. @@aut@@ |
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Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. © Springer-Verlag 2010 |
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Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. © Springer-Verlag 2010 |
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Abstract Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 μg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences. © Springer-Verlag 2010 |
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