Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.     

Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.     

A silica sands-based method for faithful analysis of microbial communities and DNA isolation from a wide range of species

A silica sands-based method has been developed to isolate high quality genomic DNAs from cells of animals, plants and microorganisms, such as Hemisalanx prognathus, Spinacia oleracea, Pichia pastoris, Bacillus licheniformis and Escherichia coli. To the best of our knowledge, no DNA isolation method has so wide application until now. In addition, this method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16s rDNA sequencing. As a result, the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit, indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities.     

A simple protocol for isolation of fungal DNA

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.     

A modified procedure for isolation of yeast mitochondrial DNA

A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances.     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

A novel method for the isolation of mycobacterial DNA

Abstract DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to −70 °C, then incubated at 65 °C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 × 10⁹) or more cells) including Mycobacterium neoaurum M. fortuitum M. phlei and M. smegmatis . Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis .     

DNA isolation protocol for seaweeds

We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the pellet. This provides a higher yield of DNA, in the range of 40 μg (Palmaria palmata) and 18 μg (Gracilaria verrucosa) from 50 mg of fresh frozen pellet.     

A PCR-based method for isolation of genomic DNA flanking a known DNA sequence

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR.     

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.     

A simple method for isolation of megabase DNA from cotton

A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning.     

碱裂解提取质粒DNA的改进

碱裂解提取质粒DNA是分子生物学实验中常用的方法,但通常方法所提取的质粒往往含有大量的RNA和其他杂质.本文适时加入较高浓度的RNA酶和适当延长冰浴时间,结果得到了几乎没有RNA和其他杂质的高纯质粒DNA,不仅达到了分子生物学实验要求,而且可用作抗原检测抗dsDNA抗体.该法操作简单、经济、实用.     

A simple method for isolation of high-molecular-weight DNA from Rhodotorula gracilis

Unsheared DNA has been isolated from Rhodotorula and Rhodosporidium yeasts using a cell-wall-digesting enzyme preparation from Paecilomyces lilacinus. Pulsed-field gel electrophoresis indicated that at least 11 chromosomes were present in Rhodot. gracilis ATCC 90950. The DNA was amenable to digestion with restriction enzymes.The authors are with the Department of Microbiology, Central Food Technological Research Institute, Mysore-570 013, India.     

A new protocol for isolation of mitochondrial DNA from cotton seedlings

A procedure to isolate mtDNA from cotton seedlings G. hirsutum and G. barbadense has been developed. The new protocol allows for the isolation of cotton mtDNA of high purity, yield and digestibility by restriction endonucleases. The success of the protocol is based on critical adjustments in the ionic strength of the homogenizing medium and washing buffer, the speed of grinding during homogenization, and the methods used for lysis of the mitochondria.     

A simplified protocol for routine total DNA isolation from salmonid fishes

An efficient total DNA isolation protocol, suitable for routine population genetic screening purposes is described. This phenol based extraction can utilize fresh, frozen or ethanol preserved tissues.