Understanding complex functional mechanisms requires the global and parallel analysis of different cellular processes. DNA microarrays have become synonymous with this kind of study and, in many cases, are the obvious platform to achieve this aim. They have already made important contributions, most notably to gene-expression studies, although the true potential of this technology is far greater. Whereas some assays, such as transcript profiling and genotyping, are becoming routine, others are still in the early phases of development, and new areas of application, such as genome-wide epigenetic analysis and on-chip synthesis, continue to emerge.

Microarray technology: beyond transcript profiling and genotype analysis

Oligonucleotide microarray (DNA chip)–based hybridization analysis is a promising new technology which potentially allows rapid and cost–effective screens for all possible mutations and sequence variations in genomic DNA. Here, I review current strategies and uses for DNA chip–based resequencing and mutational analysis, the underlying principles of experimental designs, and future efforts to improve the sensitivity and specificity of chip–based assays.

Resequencing and mutational analysis using oligonucleotide microarrays

Peptide Nucleic Acid. A Molecule with Two Identities

Selection and adjustment of proper physical parameters enables rapid DNA transport, site selective concentration, and accelerated hybridization reactions to be carried out on active microelectronic arrays. These physical parameters include DC current, voltage, solution conductivity and buffer species. Generally, at any given current and voltage level, the transport or mobility of DNA is inversely proportional to electrolyte or buffer conductivity. However, only a subset of buffer species produce both rapid transport, site specific concentration and accelerated hybridization. These buffers include zwitterionic and low conductivity species such as: d- and l-histidine; 1- and 3-methylhistidines; carnosine; imidazole; pyridine; and collidine. In contrast, buffers such as glycine, beta-alanine and gamma-amino-butyric acid (GABA) produce rapid transport and site selective concentration but do not facilitate hybridization. Our results suggest that the ability of these buffers (histidine, etc.) to facilitate hybridization appears linked to their ability to provide electric field concentration of DNA; to buffer acidic conditions present at the anode; and in this process acquire a net positive charge which then shields or diminishes repulsion between the DNA strands, thus promoting hybridization.

/pdf/electric-field-directed-nucleic-acid-hybridization-on-3qya28wwob.pdf

Electric Field Directed Nucleic Acid Hybridization on Microchips

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.

/pdf/hybridisation-based-dna-screening-on-peptide-nucleic-acid-cmqo1im746.pdf

Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays

Combining the Preparation of Oligonucleotide Arrays and Synthesis of High-Quality Primers

Modified phosphoramidite chemistry allows the generation of oligonucleotide arrays containing molecules of high purity. Such arrays are being used for experiments on the determination of expression levels in yeast and for investigations of the hybridization behaviour of modified oligonucleotides such as phosphorothioate derivatives.

New developments in oligomer array technologies

Die Erfindung betrifft ein Verfahren zur parallelen Synthese von Oligonukleotiden auf einer Alkylamino-modifizierten Matrixoberflache, dadurch gekennzeichnet, das 3-Succinat-Derivate von geschutzen Nukleosiden darauf angebracht werden und Oligonukleotidsynthese mittels automatisierter DNA-Synthese stattfindet. Die Erfindung betrifft ferner die Verwendung des bei dem Verfahren entstehenden Oligomer-Chips.

Verfahren zur kombination von paralleler oligonukleotidsynthese und darstellung von oligomer-chips

The invention concerns a process for the parallel synthesis of oligonucleotides on an alkylamino-modified matrix surface, characterized in that 3-succinate derivatives of protected nucleosides are applied thereto and oligonucleotide synthesis takes place by means of automated DNA synthesis. The invention also concerns the use of the oligomer chip formed in the process.

Process for combining parallel oligonucleotide synthesis

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