Effect of Tris-HCl Buffer on DNA Adsorption by a Variety of Soil Constituents

Saeki, Kazutoshi; Kunito, Takashi; Sakai, Masao

doi:10.1264/jsme2.me10172

Cited by 30 publications

(19 citation statements)

References 34 publications

(40 reference statements)

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“…Tris is commonly used as a biological buffer. 29–31 The effect of adding TBE in the silk/DNA mixture on DNA preservation was evaluated. The samples were stored at different temperature for 24 hours before being analyzed.…”

Section: Resultsmentioning

confidence: 99%

DNA preservation in silk

et al. 2017

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The structure of DNA is susceptible to alterations at high temperature, pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells as well as plasmid DNA was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA+filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA+PT-filter) as a route to practical utility. After air-drying and water extraction, 50–70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. The 6% silk/DNA+PT-filter samples provided improved stability in comparison to 3% silk/DNA+filter and DNA+filter for DNA preservation, with ~40% of the band intensity remaining at 37 °C after 40 days and ~10% after exposure to the UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/Borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluoresence protein (dsRed) in HEK293 cells. The high molecular weight and high content of crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA+PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

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Section: Resultsmentioning

confidence: 99%

DNA preservation in silk

et al. 2017

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“…We used distilled water instead of tris(hydroxymethyl)aminomethane hydrochloride (TrisHCl) buffer, which is widely used for experiments using DNA, because Tris molecules would affect the adsorption amount of DNA on clay minerals and humic acids (Saeki et al 2011).…”

Section: Dna Adsorption Experimentsmentioning

confidence: 99%

Strong adsorption of DNA molecules on humic acids

et al. 2011

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Analysing soil microbial communities is often hampered by DNA adsorption on soil organic compounds such as humic acids. However the role of humic acids in DNA adsorption and stability in soils remains controversial. To characterize DNA-humic acid interactions, we studied DNA adsorption on two commercially available humic acids and a soil humic acid extracted from an Andosol. Desorption of the adsorbed DNA using 4 different solutions-distilled water, 0.1 M NaCl, 0.1 M sodium phosphate buffer (pH 6.0), and 1% sodium dodecyl sulfate solution-was also studied to understand the mechanism of DNA adsorption on humic acids. Here, we show that humic acids play an important role in DNA adsorption to soils. DNA molecules were adsorbed on the humic acids, with adsorption increasing proportionally with the DNA concentrations in the solution. The adsorption on all humic acid samples was fitted with Freundlich equation, and the parameters obtained from the equation indicated a high affinity between the humic acids and DNA molecules. The total amount of DNA desorbed by the 4 solutions was less than 2% of the total DNA adsorbed on all the humic acids. The results demonstrate that DNA molecules are able to bind strongly to humic acids by ligand binding, hydrophobic interaction, aggregation, or precipitation.

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“…However, the mechanism(s) by which skim milk enhances the extraction of DNA from Andosol is (are) not clear and it is not known which constituents of skim milk are responsible for the observed effects. DNA adsorption on soil particles can be affected by organic solutes; for example, proteins coating DNA enhanced the amount of DNA adsorbed by clay (Pietramellara et al 2007); Tris buffer promoted DNA adsorption by Andosols and allophanes (Saeki et al 2011a). Therefore, we assume that protein constituents of skim milk would be involved in preventing DNA adsorption to soil particles during extraction; α-casein is a representative phosphorylated protein in milk.…”

Section: Introductionmentioning

confidence: 99%

Effect of α-casein on DNA adsorption by Andosols and by soil components

2011

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Andosols and the soil components (allophanes, humic acids, and goethite) had been autoclaved to destroy the nuclease activity of soil microflora. DNA adsorption by allophanes and Andosols was decreased by increasing the amount of α-casein added to the allophanes and to soils up to casein concentration of 5 mg ml −1 . DNA adsorption by humic acids was significantly increased by increasing the amount of α-casein up to 1.0 mg ml −1 , whereas the addition of 20 mg α-casein ml −1 completely blocked DNA adsorption. These results can explain why the addition of excess skim milk is operationally needed for effective DNA extraction from Andosols. The amount of DNA adsorbed by Andosols treated with dephosphorylated α-casein was significantly higher than that of not treated Andosols (p<0.01), thus showing an opposite behavior than α-casein addition, implying that phosphate groups of the protein can contribute to the decrease in the DNA adsorption by soil components. These results explain that the usefulness of skim milk as an additive for the extraction of DNA from Andosols is due to the presence of phosphate groups and hydrophobic components of casein, the main constituent of skim milk.

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Effect of Tris-HCl Buffer on DNA Adsorption by a Variety of Soil Constituents

Cited by 30 publications

References 34 publications

DNA preservation in silk

DNA preservation in silk

Strong adsorption of DNA molecules on humic acids

Effect of α-casein on DNA adsorption by Andosols and by soil components

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