Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- SD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Sorptive interaction between goethite and strongly reducing organic substances from anaerobic decomposition of green manures

Strongly reducing organic substances (SROS) and iron oxides exist widely in soils and sediments and have been implicated in many soil and sediment processes. In the present work, the sorptive interaction between goethite and SROS derived from anaerobic decomposition of green manures was investigated by differential pulse voltammetry (DPV). Both green manures, Astragaltus sinicus (Astragalus) and Vicia varia (Vicia) were chosen to be anaerobically decomposed by the mixed microorganisms isolated from paddy soils for 30 d to prepare different SROS. Goethite used in experiments was synthesized in laboratory. The anaerobic incubation solutions from green manures at different incubation time were arranged to react with goethite, in which SROS concentration and Fe(II) species were analyzed. The anaerobic decomposition of Astragalus generally produced SROS more in amount but weaker in reducibility than that of Vicia in the same incubation time. The available SROS from Astragalus that could interact with goethite was 0.69 +/- 0.04, 0.84 +/- 0.04 and 1.09 +/- 0.03 cmol kg(-1) as incubated for 10, 15 and 30 d, respectively, for Vicia, it was 0.12 +/- 0.03, 0.46 +/- 0.02 and 0.70 +/- 0.02 cmol kg(-1). One of the fates of SROS as they interacted with goethite was oxidation. The amounts of oxidizable SROS from Astragalus decreased over increasing incubation time from 0.51 +/- 0.05 cmol kg(-1) at day 10 to 0.39 +/- 0.04 cmol kg(-1) at day 30, but for Vicia, it increased with the highest reaching to 0.58 +/- 0.04 cmol kg(-1) at day 30. Another fate of these substances was sorption by goethite. The SROS from Astragalus were sorbed more readily than those from Vicia, and closely depended upon the incubation time, whereas for those from Vicia, the corresponding values were remarkably less and apparently unchangeable with incubation time. The extent of goethite dissolution induced by the anaerobic solution from Vicia was greater than that from Astragalus, showing its higher reactivity. (c) 2008 Published by Elsevier Ltd.

An improved method for the extraction of low molecular weight organic acids in variable charge soils

Due to specific adsorption to variable charge soils, low molecular weight organic acids (LMWOAs) have not been sufficiently extracted, even if common extractants, such as water and 0.1 M sodium hydroxide (NaOH), were employed. In this work, the method for extracting LMWOAs in soils with 0.1 M NaOH was improved for variable charge soils; e.g. 1.0 M potassium fluoride (KF) with pH 4.0 was applied as an extractant jointed with 0.1 M NaOH based on its stronger ability to change the electrochemical properties of variable charge soils by specific adsorption. With the proposed method, the recoveries of oxalic, tartaric, malic, citric and fumaric acids were increased from 83 4, 93 1, 22 2, 63 +/- 5 and 84 +/- 3% to 98 +/- 2, 100 +/- 2, 85 +/- 2, 90 +/- 2 and 89 +/- 2%, respectively, compared with NaOH alone. Simultaneously, the LMWOAs in Agri-Udic Ferrosol with field moisture were measured with a satisfactory result.

Optimization of an effective extraction procedure for the analysis of microcystins in soils and lake sediments

Microcystin analysis in sediments and soils is considered very difficult due to low recovery for extraction. This is the primary limiting factor for understanding the fate of toxins in the interface between water and sediment in both the aquatic ecosystem as well as in soils. In the present study, a wide range of extraction solvents were evaluated over a wide range of pH, extraction approaches and equilibration time to optimize an effective extraction procedure for the analysis of microcystins in soils and lake sediments. The number of extractions required and acids in extraction solutions were also studied. In this procedure, EDTA-sodium pyrophosphate solution was selected as an extraction solvent based on the adsorption mechanism study. The optimized procedure proved to be highly efficient and achieved over 90% recovery. Finally, the developed procedure was applied to field soil and sediment sample collected from Chinese lakes during bloom seasons and microcystins were determined in six of ten samples. (c) 2005 Elsevier Ltd. All rights reserved.

A solid-phase microextraction fiber coated with diglycidyloxycalix[4]arene yields very high extraction selectivity and sensitivity during the analysis of chlorobenzenes in soil

A novel fiber coated with novel sol-gel (5,11,17,23-tetra-tert-butyl-25,27-dihydroxy-26,28-diglycidyloxycalix[4]arene/hydroxy-terminated silicone oil; diglycidyloxy-C[4]/OH-TSO) was prepared for use with headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) and electron capture detection (ECD), which was applied in order to determine nine chlorobenzenes in soil matrices. Due to the improved fiber preparation, which increases the percentage of calixarene in the coating, the new calixarene fiber exhibits very high extraction selectivity and sensitivity to chlorine-substituted compounds. Various parameters affecting the extraction efficiency were optimized in order to maximize the sensitivity during the chlorobenzene analysis. Interferences from different soil matrices with different characteristics were investigated, and the amount extracted was strongly influenced by the matrix. Therefore, a standard addition protocol was performed on the real soil samples. The linear ranges of detection for the chlorobenzenes tested covered three orders of magnitude, and correlation coefficients > 0.9976 and relative standard deviations (RSD) < 8% were observed. The detection limits were found at sub-ng/g of soil levels, which were about an order of magnitude lower than those given by the commercial poly(dimethylsiloxane) (PDMS) coating for most of the compounds. The recoveries ranged from 64 to 109.6% for each analyte in the real kaleyard soil matrix when different concentration levels were determined over the linear range, which confirmed the reliability and feasibility of the HS-SPME/GC-ECD approach using the fiber coated with diglycidyloxy-C[4]/OH-TSO for the ultratrace analysis of chlorobenzenes in complex matrices.

Determination of phthalate acid esters plasticizers in plastic by ultrasonic solvent extraction combined with solid-phase microextraction using calix[4]arene fiber

Ultrasonic solvent extraction combined with solid-phase microextraction (SPME) with calix[4]arene/hydroxy-terminated silicone (C[4]/OHTSO) oil coated fiber was used to extract phthalate acid esters (PAEs) plasticizers in plastic, such as blood bags, transfusion tubing, food packaging bag, and mineral water bottle for analysis by gas chromatography (GC). Both extraction parameters (i.e. extraction time, extraction temperature, ionic strength) and conditions of the thermal desorption in a GC injector were optimized by analysis of eight phthalates. The fiber shows wonderful sensitivity and selectivity to the tested compounds. Owing to its high thermal stability (380 degreesC), the carryover effect that often encountered when using conventional fibers can be reduced by appropriately enhancing the injector temperature. The method showed linear response over two to four orders of magnitude with correlation coefficients (r) better than 0.996, and limits of detection (LOD) ranged between 0.006 and 0.084 mug l(-1). The relative standard deviation values obtained were less than or equal to 10%. bis-2-Ethylhexyl phthalate (DEHP) was the sole analyte detected in these plastics and recoveries were in the ranges 95.5-101.4% in all the samples. (C) 2004 Elsevier B.V. All rights reserved.