Ian J. Bruce

Recent developments of magnetic beads for use in nucleic acid purification

The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA. Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.

Application of magnetite and silica–magnetite composites to the isolation of genomic DNA

Magnetite and silica-magnetite composites were used as adsorbents for the isolation of genomic DNA from maize kernels. Two methods are described for the preparation of silica-magnetite composites, both of which afford higher yields of genomic DNA than when using magnetite alone, or a commerically available kit. DNA isolated using silica-magnetite was suitable for use in further applications such as polymerase chain reaction amplification and enzyme digestion.

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples.

Aims:  A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out.

Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples.

Inorganic materials for bone repair or replacement applications

In recent years, excipient systems have been used increasingly in biomedicine in reconstructive and replacement surgery, as bone cements, drug-delivery vehicles and contrast agents. Particularly, interest has been growing in the development and application of controlled pore inorganic ceramic materials for use in bone-replacement and bone-repair roles and, in this context, attention has been focused on calcium-phosphate, bioactive glasses and SiO2- and TiO2-based materials. It has been shown that inorganic materials that most closely mimic bone structure and surface chemistry most closely function best in bone replacement/repair and, in particular, if a substance possesses a macroporous structure (pores and interconnections >100 microm diameter), then cell infiltration, bone growth and vascularization can all be promoted. The surface roughness and micro/mesoporosity of a material have also been observed to significantly influence its ability to promote apatite nucleation and cell attachment significantly. Pores (where present) can also be packed with pharmaceuticals and biomolecules (e.g., bone morphogenetic proteins [BMPs], which can stimulate bone formation). Finally, the most bio-efficient - in terms of collagen formation and apatite nucleation - materials are those that are able to provide soluble mineralizing species (Si, Ca, PO(4)) at their implant sites and/or are doped or have been surface-activated with specific functional groups. This article presents the context and latest advances in the field of bone-repair materials, especially with respect to the development of bioactive glasses and micro/mesoporous and macroporous inorganic scaffolds. It deals with the possible methods of preparing porous pure/doped or functionalized silicas or their composites, the studies that have been undertaken to evaluate their abilities to act as bone repair scaffolds and also presents future directions for work in that context.

Encapsulation of superparamagnetic nanoparticles into red blood cells as new carriers of MRI contrast agents.

AIMS The half-life of superparamagnetic iron oxide nanoparticles in the bloodstream is very short since they are rapidly taken up by the reticuloendothelial system. In this article, we report the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time. MATERIALS & METHODS Newly synthesized and commercially available nanoparticles were evaluated for the encapsulation into red blood cells through the transient opening of membrane pores by controlled hypotonic dialysis and successive isotonic resealing and reannealing of cells. RESULTS Commercial superparamagnetic iron oxide nanoparticles (SHU 555A, AMI 227 and PMP-50) dextran or carboxydextran coated can be successfully loaded into red blood cells; similarly, some of the new nanomaterials, such as Np-1 nanoparticles dispersed in the Disperbyk®-190 agent, can be efficiently encapsulated into red blood cells. CONCLUSION A careful consideration of magnetic nanoparticles parameters, such as size, synthesis protocols, coating and/or dispersant agents, is required in order to obtain efficient loading through the cell membrane pores.

PCR detection of genetically modified soya and maize in foodstuffs.

The detection of genetically modified foodstuffs is becoming both a food sales and legal necessity. This study reports a rapid DNA extraction/PCR-based method for the detection of genetically modified soya (GMS) and maize (GMM) in mixed samples of transgenic and unmodified soybeans and maize kernels, and a variety of processed samples including soya flour, soya protein isolates, extruded defatted soya, acid- and alcohol-precipitated soya concentrates, soya lecithin, maize grits, seasoned corn puffs and salted corn chips. The presence of GMS DNA was determined with two pairs of primers directed towards different GMS target sequences and GMM by one primer pair. In addition, a multiplex PCR reaction which utilises an internal positive control was developed for both genetically modified organisms (GMOs). Results indicated that the methods are sensitive and specific enough to detect GMS down to a level of 0.01% dry weight in single-product PCRs and 0.1% in multiplex PCRs and GMM down to 0.001% dry weight in single-product PCRs and 0.01% in multiplex PCR. The methods are considered to represent a viable route for the commercial detection of GMS and GMM in foodstuffs.

Synthesis of fluorescently labelled oligonucleotides and nucleic acids

The demand for and application of fluorescently labelled nucleic acid materials are growing, driven by ventures such as the Human Genome Mapping Project and the advent of DNA-based clinical diagnostics. This article surveys the strategies that are currently available for conjugation of fluorescent molecules to oligonucleotides and nucleic acids, and demonstrates how the labelled materials have been used in new and innovative assays for probing nucleic acid structure and properties.

New approaches to the isolation of DNA by ion-exchange chromatography

The performance of different anion-exchange media have been compared for the isolation of plasmid DNA and genomic DNA from bacterial cells and human whole blood. Whatman DEAE-Magarose, based on an agarose bead containing a paramagnetic component, has been compared with prepacked gravity-flow columns containing a derivatised silica matrix. In each case the DNA isolation at various scales of operation was similar both in terms of yield and quality. The magnetic susceptibility of DEAE-Magarose is very high, facilitating the use of this separation technique for rapid flexible batch chromatographic processes, a limitation of the prepacked column techniques.