Salicylhydroxamic acid functionalized affinity membranes for specific immobilization of proteins and oligonucleotides.

Immobilization of proteins and other biological macromolecules on solid supports is a method suitable for purification or screening applications in life science research. Prolinx, Inc. has developed a novel chemical affinity system that can be used for specific immobilization of proteins and other macromolecules via interaction of two small synthetic molecules, phenyldiboronic acid (PDBA) and salicylhydroxamic acid (SHA). This report describes immobilization applications of activated microporous membranes that have been functionalized with SHA derivatives. These SHA-membranes exhibit high capacity and specificity for binding of PDBA-labeled nucleic acids and proteins. Conjugation of active protein with PDBA is performed in solution independent of the immobilization step on SHA membranes. The resulting PDBA-protein conjugate is immobilized directly without purification and retains biological activity. PDBA conjugates may also be released from these SHA-affinity membranes in a controlled manner. Capture and release of PBA-modified oligonucleotides is also demonstrated. SHA-membranes can be used as surfaces for microarrays, and are therefore compatible with high-throughput analyses. These properties make them useful for development of numerous preparative or screening applications.

A rapid method for manual or automated purification of fluorescently labeled nucleic acids for sequencing, genotyping, and microarrays.

Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids. To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors. Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation. Common methods for purifying reaction products involve several steps and require processes that are not easily automated. Prolinx, Inc. has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated. The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors. These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow. RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes. RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization. The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids.

Use of a small molecule-based affinity system for the preparation of protein microarrays.

This chapter describes a method for preparing protein microarrays, using a small-molecule, chemical affinity system.