Effects of humic substances on fluorometric DNA quantification and DNA hybridization

Bachoon, Dave S.; Otero, Ernesto; Hodson, Robert E.

doi:10.1016/s0167-7012(01)00296-2

Cited by 50 publications

(35 citation statements)

References 23 publications

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“…These factors may have resulted in artificially high estimates of initial DNA yields based on PicoGreen analysis and exaggerated DNA losses upon purification (Carrigg et al 2007). Although humic acids are known to interfere with PicoGreen at high concentrations (Bachoon et al 2001), PicoGreen can be used on dilute samples in which small amounts of DNA are still present and humic acid interference is negligible (Sandaa et al 1998;Stark et al 2000). Our results indicated that DNA recovery was little affected by humic acid co-extraction; DNA recovery with M1 was found to be 46%À69% (Table 1), which is within the expected range based on the literature values (Robe et al 2003).…”

Section: Discussionsupporting

confidence: 84%

The effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods

Xiong

Xie

Wang

et al. 2014

Journal of Freshwater Ecology

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We evaluated the effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods. DNA yields and the number of ribotypes differed significantly by depth. The mean yields at the depth interval of 0À8 cm from the two extraction methods were both higher than those within the depth range of 10À44 cm. Significant differences were also observed between the two methods in DNA yields from the deeper (10À44 cm) samples. An analysis of denaturing gradient gel electrophoresis (DGGE) profiles demonstrated that the choice of the DNA extraction method has a profound effect on the bacterial community profiles generated, which was reflected in the number of bands or ribotypes detected from samples from all depths. Higher DNA yield did not lead to a higher diversity of phylotypes. In almost all cases, extraction method 2 (M2, soil DNA test kit) resulted in a greater diversity of phylotypes compared to method 1 (M1, liquid nitrogen/cetyl trimethylammonium bromide extraction). Moreover, notable changes in bacterial diversity were detected in the uppermost layers (0À5 cm). The results of canonical correspondence analysis illustrated that the DNA extraction method did not affect the evaluation of the relationship between bacterial community structure and environmental variables in lake sediments at multiple depths, and the differences in community structure in the two extraction methods were both related to the same environmental variables (pH, total phosphorus concentration) and depth. Vertically, the community structure of sediment layers formed several separate clusters along the depth gradient. This study expands our understanding of the depth-related microbial community structure in lake sediments.

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Section: Discussionsupporting

confidence: 84%

The effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods

Xiong

Xie

Wang

et al. 2014

Journal of Freshwater Ecology

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“…This measure is depicted in the upper part of figure 1. Figure 1 As reported in the literature [20,31,32,34,35], the diphenylamine and Hoechst 33258 ® based methods are only able to detect DNA, whereas the other tested protocols can also be used to quantify RNA.…”

Section: Sensitivity Of the Methodsmentioning

confidence: 87%

“…The nucleic acid determination was performed as described by Bachoon et al [34] with some modifications. Hoechst solution was fresh made by adding 4ml Hoechst 33258 ® (0.5µg/ml) to 5ml 10x TNE buffer (1M NaCl, 10mM EDTA, 0.1M Tris-HCl pH7.4) and 41ml MiliQ-H 2 O.…”

Section: Fluorescence Detection With Hoechst 33258 ®mentioning

confidence: 99%

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Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Mey

Lequeux

Maertens

et al. 2006

Analytical Biochemistry

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Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. Five current methods for nucleic acid quantification have been compared: 1) UV absorbance spectroscopy at 260nm, 2) Colorimetric reaction with the orcinol reagent, 3)Colorimetric reaction based on diphenylamine, 4) Fluorescence detection with reagent Hoechst 33258 and 5) Fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used as well as two different types of yeast RNA and a mixture of equal quantities of DNA and RNA.We can conclude that for nucleic acid quantification a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with reagent Hoechst 33258 ® is a sensitive and precise method for DNA quantification if the G+C content is lower than 50%. In addition this method allows quantification of very low levels of DNA (ng-scale). Moreover, the samples can be crude cell extracts. Also UV absorbance at 260nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids have to be measured.

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“…This particular method was employed to reduce the co-extraction of polymerase-chain-reaction (PCR) inhibiting agents. Analysis of DNA in the samples was still hampered by the co-extraction or co-purification of humic substances (Bachoon et al, 2001). Humic acids are chemically complex and polydisperse mixtures of microbiologically decomposed and transformed organic materials.…”

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confidence: 99%

Causes and Possible Solutions to the Middle East Terrorism

Al-Thagafi¹

2008

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The public reporting burden for this collection of information is estimated to average 1 hour par response, including the time for reviewing instructions, searching existing data sources, gathering and meintaining the date needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of informaton, including suggestions for reducing the burden, to Department of Defene, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondlents should be aware that notwithstanding eny other provision of law, no person shell be subject to any penalty for failing to comply with a collection of Information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY)2. REPORT TYPE 1 3. DATES COVERED (From -To) 30-11-2007 Final Technical Report Cooney, Michael J. 6t. WORK UNIT NUMBERLiaw, Bor Yann Masutani, Stephen M. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) S. PERFORMING ORGANIZATION University of Hawaii REPORT NUMBER2530 Dole Street, Sakamaki D200 Honolulu, HI 96822 SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S)Office of Naval Research ONR Regional Office Seattle 1107 NE 45th Street, Suite 350 SPONSOR/MONITOR'S REPORTSeattle, WA 98105-4631 NUMBER(S) DISTRIBUTION/AVAILABILITY STATEMENTApproved for public release; distribution is unlimited. SUPPLEMENTARY NOTESIn addition to the primary text of the Final Technical Report (106 pages), the report has three Appendices with 80 pages total. ABSTRACTThis report covers efforts by the Hawaii Natural Energy Institute of the University of Hawaii under the ONR-funded HEET Initiative that addresses critical technology needs for exploration/utilization of seabed methane hydrates and development/testing of advanced fuel cells and fuel cell systems. Methane hydrates work included: laboratory and analytical investigations of hydrate destabilization phenomena; characterization of the microbial community in marine hydrate beds; development of models to predict the fate of methane released from the seafloor into the water column; and the promotion of international R&D partnerships. In the fuel cell area, accomplishments included: addition of two fuel cell test cells to the Hawaii Fuel Cell Test Facility; improved capabilities for fuel/oxodizer impurity testing; and improved performance of the dynamic HiL test stand. Under the hydrogen production area, efforts were focused on sulfur removal from fuel gas and construction of a reformer system for seafloor methane work. Further work was also carried out with novel fuel cell concepts including biocarbons and bio-fuel cells. SUBJECT TERMS Executive SummaryThis report summarizes work conducted under Grant Number N00014-06-1-0086, the Hawaii Energy and Environmental Technologies (HEET) Initiative, funded through the Office of...

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Effects of humic substances on fluorometric DNA quantification and DNA hybridization

Cited by 50 publications

References 23 publications

The effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods

The effect of depth on microbial communities from mesotrophic lake sediments as measured by two DNA extraction methods

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Causes and Possible Solutions to the Middle East Terrorism

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