Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

Background: We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs).

Methods: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers.

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop.

Regulation and expression of multidrug resistance (MDR) transcripts in the intestinal epithelium.

A paucity of information exists on the regulation of gene expression in the undifferentiated intestine. The intestinal epithelium is one of the few normal tissues expressing the multidrug resistance (MDR) genes that confer the multidrug resistant phenotype to a variety of tumours. Expression of mdr1a has been observed in the primitive rat intestinal epithelial cell line, IEC-18.

Elimination of palmitoylation sites in the human dopamine D1 receptor does not affect receptor-G protein interaction.

We have eliminated putative palmitoylation sites in the carboxyl tail of the human dopamine D1 receptor by replacing the two cysteine residues with alanines either separately or together. The wild type and the three mutated dopamine D1 receptors were stably expressed in baby hamster kidney cells and characterized to detect any resulting alterations in receptor-G protein interactions. The three mutant dopamine D1 receptors retained the same proportion of high affinity state for agonists as wild type receptors and also no difference was observed in the stimulation of adenylyl cyclase activity.