Adsorption and binding of AmpliTaq® DNA polymerase on the clay minerals, montmorillonite and kaolinite

Vettori, Cristina; Calamai, Luca; Yoder, M.; Stotzky, G.; Gallori, Enzo

doi:10.1016/s0038-0717(98)00162-x

Cited by 27 publications

(12 citation statements)

References 29 publications

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“…The degradation of Bt proteins, as well as of other proteins, peptides, amino acids, DNA, and other biomolecules, in soil depends largely on the level of microbial activity (e.g., Stotzky 1985, 1986;Dashman and Stotzky 1986;Stotzky 1986;Khanna and Stotzky 1992;Gallori et al 1994;Vettori et al 1996Vettori et al , 1999Palm et al 1996;Koskella and Stotzky 1997;Crecchio and Stotzky 1998a;Tapp and Stotzky 1998 Fig. 3 Persistence of Cry3Bb1 protein, as determined by ELISA, in Kitchawan soil amended to 3 or 6% (vol./vol.)…”

Section: Discussionmentioning

confidence: 99%

“…israelensis (active against Diptera) are rapidly adsorbed and bound on clay minerals and humic substances, which renders them less available for biodegradation, but their insecticidal activity is retained (e.g., Stotzky 1990, 1992;Tapp et al 1994;Stotzky 1995a, b, 1998;Koskella and Stotzky 1997;Stotzky 1998a, 2001;Lee et al 2003;Stotzky 2004). Other proteins, peptides, amino acids, DNA, and viruses are also protected against microbial degradation and inactivation when bound on such surfaceactive particles (e.g., Stotzky 1985, 1986;Stotzky 1986Stotzky , 2004Dashman and Stotzky 1986;Khanna and Stotzky 1992;Gallori et al 1994;Vettori et al 1996Vettori et al , 1999Crecchio and Stotzky 1998b;Calamai et al 2000;Lozzi et al 2001;Crecchio et al 2005). Moreover, repeated and large-scale use of transgenic Bt plants could result in the accumulation and persistence of plant-produced Bt proteins in soil (e.g., Stotzky 1995a, 1998;Crecchio and Stotzky 1998a).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cry3Bb1 protein from Bacillus thuringiensis in root exudates and biomass of transgenic corn does not persist in soil

Icoz

Stotzky

2007

Transgenic Res

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The Cry3Bb1 protein, insecticidal to the corn rootworm complex (Diabrotica spp.), of Bacillus thuringiensis (Bt) subsp. kumamotoensis was released in root exudates of transgenic Bt corn (event MON863) in sterile hydroponic culture (7.5 +/- 1.12 ng/ml after 28 days of growth) and in nonsterile soil throughout growth of the plants (2.2 +/- 0.62 ng/g after 63 days of growth). Kitchawan soil, which contains predominantly kaolinite (K) but not montmorillonite (M), was amended to 3 or 6% (vol./vol.) with K (3K and 6K soils) or M (3M and 6M soils) and with 1, 3, 5, or 10% (wt./wt.) of ground biomass of Bt corn expressing the Cry3Bb1 protein and incubated at 25 +/- 2 degrees C at the -33-kPa water tension for 60 days. Soils were analyzed for the presence of the protein every 7 to 10 days with a western blot assay (ImmunoStrip) and verified by ELISA. Persistence of the protein varied with the type and amount of clay mineral and the pH of the soils and increased as the concentration of K was increased but decreased as the concentration of M was increased. Persistence decreased when the pH of the K-amended soils was increased from ca. 5 to ca. 7 with CaCO(3): the protein was not detected after 14 and 21 days in the pH-adjusted 3K and 6K soils, respectively, whereas it was detected after 40 days in the 3K and 6K soils not adjusted to pH 7. The protein was detected for only 21 days in the 3M soil and for 14 days in the 6M soil, which were not adjusted in pH. These results indicate that the Cry3Bb1 protein does not persist or accumulate in soil and is degraded rapidly.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cry3Bb1 protein from Bacillus thuringiensis in root exudates and biomass of transgenic corn does not persist in soil

Icoz

Stotzky

2007

Transgenic Res

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“…Adsorption data were also used to conduct a Scatchard plot analysis (39) of DNA binding to kaolinite particles.…”

mentioning

confidence: 99%

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

Yankson

Steck

2009

Appl Environ Microbiol

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We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, g quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6,21,22,24,37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7,21,26,34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7,10,20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment-clay that contains humic substances-and enumerating a specific target sequence from the crude extract.Kaolinite clay: DNA binding. To determine whether kaolinite would be a suitable clay type for use in optimizing DNA desorption protocols, the nature of DNA binding to pure kaolinite (KGa-1b) (4) (Clay Minerals Society; Chantilly, VA) was determined from binding isotherms. The DNA used was a 1,040-bp region of the gfp gene (GC content, ϳ38%) PCR amplified from pGFPmut3.1 (3, 9, 14) by using primers specific for this gene (5Ј-ACTGGAAAGCGGGCAGTG and 5Ј-AAA CGCGCGAGACGAAAGGG). Kaolinite was sterilized by gamma irradiation by exposure to Cobalt-60 until 70 kGy was delivered (Radiation Science and Engineering Center, Penn State University, University Park, PA), a dose sufficient to kill all living microbes (25) and to directly and indirectly damage DNA (18,30).By use of the PCR-generated gfp DNA fragment, adsorption data were fit to the Langmuir (33) and Freundlich (29) models ...

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“…These results indicate that the Cry3Bb1 protein does not persist or accumulate in soil and is degraded rapidly. In addition to the increase in microbial activity in soils with a pH near neutrality, adsorption of Cry1Ab, Cry3Aa, and Cry4 proteins on clays decreased with an increase in pH [10,61,62,72,118], which also rendered a portion the proteins more susceptible to biodegradation. Similarly, Wang et al [119] reported that the Cry1Ab protein from biomass of Bt rice degraded faster in an alkaline soil (half-life of 11.5 days) than in an acidic soil (half-live of 34.3 days).…”

Section: Release and Fate Of Bt Toxins In Root Exudates Of Transgenicmentioning

confidence: 99%

“…If confirmed, the risks to the environment of such uptake will need to be evaluated. Furthermore, although HGT among microorganisms has been demonstrated in soil microcosms [70,74,76,78,86,90,94,95,97,99,100,106,112,118,[196][197][198][199][200][201], and the cry1Ab gene has been shown to be transported in soil and waters, there is scant evidence for the uptake of cry genes by plants and other organisms from the environment [202]. Donnarumma et al studied the possible transfer of genes from Btk to the indigenous Bacillus spp.…”

Section: Uptake Of Bt Cry Proteins By Plants In Soil and In Hydroponimentioning

confidence: 99%

Fate and Effects in Soil of Cry Proteins from Bacillus thuringiensis: Influence of Physicochemical and Biological Characteristics of Soil

Saxena¹,

Pushalkar

Stotzky³

2013

TOTNJ

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Bacillus thuringiensis (Bt) is a useful alternative or supplement to synthetic chemical pesticides in agriculture, forest management, and control of mosquitoes and some other biting insects. When modified Bt cry genes are inserted into a plant species (e.g., corn, cotton, potato, canola, rice), the plant expresses active larvicidal proteins in its tissues. The toxins continue to be synthesized during growth of the plants, making the plant toxic to various insect pests throughout their life or as biomass incorporated into soil. If production exceeds consumption, inactivation, and degradation, the toxins could accumulate to concentrations that may enhance the control of target pests or constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects (e.g., pollinators, predators and parasites of insect pests), and other animal classes. The accumulation and persistence of the toxins could also result in the selection and enrichment of toxin-resistant target insects. Persistence is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays and humic substances) and, thereby, rendered more resistant to biodegradation while retaining toxic activity. Moreover, major problem we face today is of "Molecular pharming" that utilizes transgenic plants and animals for production of pharmaceuticals and chemicals for their use in human beings and industries respectively. Their release to the environment, especially to soil and potentially to waters of the pharmaceutical and industrial products of transgenic plant and animal "pharms" could pose a hazard to the environment. In contrast to the products of most transgenic plants currently available commercially (e.g., the insecticidal proteins from subspecies of Bt) that primarily target insects and other pests. These "pharms" are being genetically engineered to express products for use primarily in human beings. Consequently, these products constitute a class of compounds that is seldom found in natural habitats and that primarily target "higher level" eukaryotes. Hence, they are xenobiotics with respect to the environment, and their persistence in and effects on the environment have not been adequately studied and sober risk assessments on a case-bycase basis must be made before major releases of such transgenic organisms.

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Adsorption and binding of AmpliTaq® DNA polymerase on the clay minerals, montmorillonite and kaolinite

Cited by 27 publications

References 29 publications

Cry3Bb1 protein from Bacillus thuringiensis in root exudates and biomass of transgenic corn does not persist in soil

Cry3Bb1 protein from Bacillus thuringiensis in root exudates and biomass of transgenic corn does not persist in soil

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

Fate and Effects in Soil of Cry Proteins from Bacillus thuringiensis: Influence of Physicochemical and Biological Characteristics of Soil

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