Methods for microbial DNA extraction from soil for PCR amplification

Yeates, Christine; Gillings, Michael R.; Davison, Annette; Altavilla, Nanda; Veal, Duncan

doi:10.1251/bpo6

Cited by 243 publications

(171 citation statements)

References 23 publications

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“…Extraction of DNA from the homogenised fecal samples was carried out, using the FastPrep system (Bio101, La Jolla, CA, USA) with modifications as described previously (Yeates et al, 1998). Briefly, 0.5 ml of homogenized fecal sample was placed in a screw-cap tube with a single 5 mm glass bead and 0.3 g each of 150-212 and 472-600 mm glass beads (Sigma-Aldrich, St Louis, MO, USA).…”

Section: Dna Extractionmentioning

confidence: 99%

The immediate environment during postnatal development has long-term impact on gut community structure in pigs

2008

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The gastrointestinal tract of mammals contains a complex microbial community that influences numerous aspects of health and development. It is postulated that establishment of this community during early life has long-term consequences on the health status of adults. Potential influences on colonization are expected to include environmental microbes, diet and the developmental changes of the host. Denaturing gradient gel electrophoresis was used to follow the individual community dynamics of 24 piglets over the period of 3-36 days after birth. The community of piglets older than 31 days was inferred to show high stability relative to the first 28 days post birth. The stable day 36 community showed significant correlation between cohabiting piglets, but not between siblings. This cohabitation effect was not observable in 1-or 2-week-old piglets but was strongest at either 3 or 4 weeks post birth. The onset of this change after 2 weeks is predicted to be after the development of key induction elements of the immune system and before significant levels of piglet sIgA were observable (4 weeks). The outcome is altered community dynamics that result in significant similarity between the stable communities that develop in cohabiting pigs. We conclude that for a finite period in their development, the outcome of gut colonization in piglets is greatly influenced by the immediate environment. The implication is that mammals have a developmental window, in which the developing host-gut microbiota interaction will be simultaneously more amenable to engineering and more susceptible to disturbance.

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Section: Dna Extractionmentioning

confidence: 99%

The immediate environment during postnatal development has long-term impact on gut community structure in pigs

2008

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“…It remains to be determined whether or not such measures will correlate in any way with disease-suppressive soil environments, or whether specific microbes or suites of microbes will be consistently and strongly linked to disease suppression. Further, there are technical and taxonomic issues that need to be addressed and standard extraction protocols that need to be developed (Pankhurst et al 1996;Yeates et al 1998;Bürgmann et al 2001). Without proper evaluation, procedures may be used that preferentially extract DNA from certain taxonomic groups, leading to inaccurate characterization of populations (Martin-Laurent et al 2001).…”

Section: Measuring Diversitymentioning

confidence: 99%

Fungal plant pathogens and soil biodiversity

Reeleder¹

2003

Can. J. Soil. Sci.

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The role of biodiversity as it affects the control of soil-borne fungal pathogens is discussed. Soil-borne fungal plant pathogens have often proven difficult to manage with conventional methods of disease control. Nonetheless, researchers have characterized several naturally occurring “disease-suppressive” soils where crop loss from disease is less than would otherwise be expected. Suppressive soils can also result from the incorporation of various amendments into soil. In most cases, disease control in such soils has been shown to be biological in nature; that is, soil organisms appear to directly or indirectly inhibit the development of disease. Increased knowledge of the identity and functioning of these organisms may support the development of techniques that can be used to develop suppressiveness in soils that are otherwise disease-conducive. Populations of pathogens themselves have been shown to exhibit considerable genetic diversity; the ability of populations to respond to disease control measures should be considered when developing a management strategy. New molecular techniques can be exploited to better characterize soil communities, including the pathogens themselves, as well as community responses to various disease control options. The contributions of Canadian researchers to these areas are discussed and models for further study are proposed. Key words: Biocontrol, molecular technologies, functional diversity, integrated pest management

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“…In order to assess the efficiency in removing proteins during the purification step, we acquired the absorbance values at 260 nm (characteristic to DNA) and 280 nm (characteristic to proteins). The resulting absorbance ratio, A260/A280 (with 1.8 generally indicating high purity) is indicative of the relative amount of protein contaminants in DNA samples 34 . The A260/A280 ratio was 0.86 ± 0.09 before purification, indicating a strong presence of proteins in the loading solution.…”

Section: Resultsmentioning

confidence: 99%

Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes

et al. 2016

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Nucleic acid-based biosensors have enabled rapid and sensitive detection of pathogenic targets; however, these devices often require purified nucleic acids for analysis since the constituents of complex biological fluids adversely affect sensor performance. This purification step is typically performed outside the device, thereby increasing sample-to-answer time and introducing contaminants. We report a novel approach using a multifunctional matrix, nanoporous gold (npAu), which enables both detection of specific target sequences in a complex biological sample and their subsequent purification. The np-Au electrodes modified with 26-mer DNA probes (via thiolgold chemistry) enabled sensitive detection and capture of complementary DNA targets in the presence of complex media (fetal bovine serum) and other interfering DNA fragments in the range 50 to 1500 base pairs. Upon capture, the non-complementary DNA fragments and serum constituents of varying sizes were washed away. Finally, the surface-bound DNA-DNA hybrids were released by electrochemically cleaving the thiol-gold linkage and the hybrids were iontophoretically eluted from the nanoporous matrix. The optical and electrophoretic characterization of the analytes before and after the detection-purification process revealed that low target DNA concentrations (80 pg/µL) can be successfully detected in complex biological fluids and subsequently released to yield pure hybrids free of polydisperse digested DNA fragments and serum biomolecules. Taken together, this multifunctional platform is expected to enable seamless integration of detection and purification of nucleic acid biomarkers of pathogens and diseases in miniaturized diagnostic devices.

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Methods for microbial DNA extraction from soil for PCR amplification

Cited by 243 publications

References 23 publications

The immediate environment during postnatal development has long-term impact on gut community structure in pigs

The immediate environment during postnatal development has long-term impact on gut community structure in pigs

Fungal plant pathogens and soil biodiversity

Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes

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