DNA extraction from hair shafts of wild Brazilian felids and canids

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.

Solid-phase peptide synthesis in highly loaded conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CTAB methods for DNA extraction of sweetpotato for microsatellite analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of sugar cane bagasse as solid phase extractor for cadmium determination by FAAS

The present paper describes the use of sugar cane bagasse as solid phase extractor for cadmium determination after complexation of the analyte with ammonium diethyldithiophosphate (ADDP) and sorption of the Cd-DDP complexes on the solid support. The concomitants were separated using a flow injection analysis (FIA) system coupled to flame atomic absorption spectrometry (FAAS) for determination. The main parameters such as ADDP concentration, acid medium, flow rate, reaction coil length, and reaction time were investigated.The results obtained with HNO3 showed good accuracy and precision. The enhancement factor was 20.5 times for a 120-second preconcentration time, and the analytical frequency was 25 determinations per hour. The calibration curve was linear over the concentration range of 1-40 mu g L-1 Cd with a LOD of 0.697 mu g L-1 Cd and a relative standard deviation of 0.96% after 12 successive measurements of 30 mu g L-1 Cd.The proposed method was evaluated for the FIA-FAAS analysis of certified reference materials (tomato leaves, spinach leaves, and bovine liver) and Cd-spiked foods (shrimp, sardine, tuna, chicken liver and bovine liver). Good recoveries (80.0-97.1%) for the Cd-spiked samples and certified reference materials were obtained. The results of bagasse-packed minicolumns were compared with Si-C,8 packed minicolumns. The F-test was applied between Si-C-18/Bagasse minicolumns, Si-C-18/certified values, and bagasse/certified values. It was found that the results were in agreement with the certified values at a 95% confidence level.

Comparative investigation of the cleavage step in the synthesis of model peptide resins: Implications for N-alpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the N-alpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (All, DRVYIHPF) and its [Gly(8)]-All analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%.) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.

Solvent free esterification reactions using Lewis acids in solid phase catalysis

A clean, efficient and fast method for esterification reactions for sterically (biodiesels) or otherwise inactive (aromatic) precursors was developed, using catalysts supported in a solid phase under solvent free conditions, and whose reactions can be promoted by MW irradiation. (c) 2006 Elsevier B.V. All rights reserved.

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L-1 of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and continued by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1: 10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1: 1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application. (c) 2007 Elsevier B.V. All rights reserved.

Determination of tetramethylthiuram disulfide (thiram) for residual analysis in food using spectrophotometry coupled with a solid-phase reactor (SPR) in a flow system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Use of Headspace Solid-Phase Microextraction (HS-SPME) to Assess the Quality and Stability of Fruit Products: An Example Using Red Mombin Pulp (Spondias purpurea L.)

The present study aimed to evaluate the volatiles profile of red mombin (Spondias purpurea) pulp and its powder produced by spray-drying (SD) as an example to show utility of headspace solid-phase microextraction (HS-SPME) in the analysis of parameters such as the quality and stability of fruit products. Volatiles profiles of the pulp were identified by gas chromatography-mass spectrometry (GC-MS), quantified by gas chromatography-flame ionization detector (GC-FID) and compared to the profile of the powder stored at 0, 60 and 120 days in plastic (PP) or laminated packages (LP). The results showed that the technique was able to identify 36 compounds in the red mombin pulp, 17 out of which have been described for the first time in this fruit, showing that red mombin fresh pulp appears to be unique in terms of volatiles composition. However, only 24 compounds were detected in the powder. This decrease is highly correlated (r(2) = 0.99), at least for the majority of compounds, to the degree of volatility of compounds. Furthermore, the powder stored in PP or LP showed no statistical differences in the amounts of its components for a period of 120 days of storage. Finally, this work shows how HS-SPME analysis can be a valuable tool to assess the quality and stability of fruit products.

Improving DNA extraction quality by the salting-out method to prepare blood samples for real-time PCR.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)