Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast (original) (raw)
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Applied and Environmental Microbiology
A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 pl from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 ,ug/,ul, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibition assays with DNA-transforming enzymes. Restriction endonucleases were inhibited at humic acid concentrations of 0.5 to 17.2 ,ug/ml for the commercial product and 0.8 to 51.7 1g/ml for the coextracted humic acids. DNase I was less susceptible (MIC of standard humic acids, 912 ,Ig/ml), and RNase could not be inhibited at all (MIC, >7.6 mg/ml). High inhibitory susceptibilities for humic acids were observed with Taq polymerase. For three Taq polymerases from different commercial sources, MICs were 0.08 to 0.64 ILg of the standard humic acids per ml and 0.24 to 0.48 ,ug of the coextracted humic acids per ml. The
The effects of humic substances on DNA isolation from soils
PeerJ, 2020
Background Humic substances (HS) are compounds with a complicated structure, present in the humus soil layer, water, lake sediments, peat, brown coal and shales. Due to their similar physicochemical properties to DNA, they may have an adverse effect on the subsequent use of the isolated material. The main aim of this research was to examine the effect of HS on DNA isolation depending on the soil type and land use, taking into account the spectroscopic full characteristics of HS fractions. Methods The research was conducted on eight types of soil sample. Soils represented the most important Soil Reference Groups for temperate climates: Fluvisols, Regosols, Cambisols, Arenosols, Histosols and Luvisols. Soil samples were also collected from areas diversified in terms of use: arable land, grassland and forest. The extraction of HS fractions was performed using the procedure recommended by the International HS Society. The fractional composition of HS was characterized by UV–Vis and fluo...
Journal of Applied Microbiology, 2007
Aim: To establish a rapid, improved soil environmental DNA extraction and purification protocol. Methods and Results: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. Conclusions: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. Significance and Impact of the Study: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.
Interaction of humic acids with human DNA: Proposed mechanisms and kinetics
ELECTROPHORESIS, 2008
Human DNA quantification by quantitative real-time PCR (QRT-PCR) has gained great importance in forensic DNA and ancient DNA studies. However, in such samples, DNA quantification is impaired by the frequently present humic acid (HA). We have previously shown that the addition of synthetic HA inhibits QRT-PCR. In this study we investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT-PCR inhibition. In QRT-PCR with pure human DNA and no HA added, V MAX was 40. With DNA sample containing 4 mg/mL of HA, V MAX was 30.30 while the addition of extra Taq polymerase to the same sample changed V MAX into 38.91, amplifying between 80 and 90% of input DNA. The K M /V MAX ratio in all the samples remained constant, indicating that the mechanism of HA inhibition of QRT-PCR is uncompetitive by nature. Moreover, HA shifts the human DNA melting temperature point (T m ) from 75 to 877C and inhibits DNase Imediated DNA cleavage, most probably affecting the enzyme's activity.
Effects of humic substances on fluorometric DNA quantification and DNA hybridization
Journal of Microbiological Methods, 2001
DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA Ž . amended with humic substances 20-80 ngrml using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ngrml of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic Ž . substances reduced the binding of DNA onto the nylon membrane. Low amounts -2.0 mg of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 mg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes.
Nucleic Acids Research, 2003
DNA quanti®cation of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quanti®cation is impaired by humic acids (HA). Due to its lack of speci®city and sensitivity, UV spectrophotometry cannot be applied. Consequently,¯uorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25-and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric,¯uorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner ®lter effect, collisional quenching and binding of SG to HA. As to the latter ®nding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A signi®cant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modi-®ed SG assay and the corresponding evaluation methods were veri®ed by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay.
Assessing PCR Inhibition from Humic Substances
2010
Inhibition remains the greatest methodological challenge in molecular analysis of buried biological remains. Inhibitory compounds associated with soil environments comprise primarily of humic acids and fulvic acids, collectively referred to as humic substances. We examined the sensitivity of 13 DNA polymerases to both humic acids (11ng-110 g) and fulvic acids (9.4ng-94 g) and the concentration at which successful amplification can be achieved. This research identified that all 13 DNA polymerases tested exhibited inhibition with varying concentrations of humic acids and that 5 out of the 13 DNA polymerase tested exhibited inhibition with varying concentrations of fulvic acid. The most tolerant DNA polymerase to inhibition due to the presence of humic and fulvic acids is pfu DNA polymerase followed by KlenTaq ® LA DNA polymerase and RealTaq DNA polymerase that were both only inhibited by 11 g and 110 g of humic acids. In addition, we present the use of size exclusion chromatography to remove small molecular weight humic substance, dramatically increasing the success of molecular analysis on material associated with burial. This research has implications to the fields of environmental microbiology, soil science, forensic science and archaeological science.
African Journal of …, 2010
Direct extraction of DNA from soils is a useful way to gain genetic information on the soil source. However, DNA extraction from soils, especially forest soils, may be contaminated by humic substances due to their similar physical and chemical characteristics to soil. Even commercial soil DNA extraction kits fail to retrieve DNA from these soils. Using the potential changes of specific charge of DNA and humic substances in a pH solution, we performed two electrophoreses in different pH buffers to eliminate the interfering substances. The method produced high quality soil DNA, which is applicable for PCR amplification.
Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid
Croatian medical journal, 2005
To investigate the dose-response effect of humic acid (HA) on the quantitative real time polymerase chain reaction (QRT-PCR) inhibition and the efficiency of Taq polymerase increment in preventing inhibition by HA in DNA extracted from ancient bones. DNA was isolated from bone samples and DNA quantification was conducted with the real-time 5' exonuclease detection assay (TaqMan), using the ABI PRISM 7000 instrument. The addition 10-75 ng of synthetic HA inhibited QRT-PCR, whereas the addition of 100 ng of synthetic HA completely inhibits QRT-PCR. The addition of 1.25 Unit (U) of Taq polymerase per assay appeared to be the optimum amount in overcoming the HA inhibition. The best results were obtained when crude DNA extracts containing humic substances were quantified by QRT-PCR with the addition of 1.25 Unit (U) of extra Taq polymerase per assay. The modified procedure with increased Taq polymerase concentration should allow more effective QRT-PCR analysis in samples containing HA.