Quantitative discrimination of 16 S rRNA genes of Dehalococcoides species by MagSNiPer, a quantitative single-nucleotide-polymorphism genotyping method.

Previously we developed MagSNiPer, an SNP (single nucleotide polymorphism) genotyping method. In the present paper we show development of an automated system for MagSNiPer, namely MagSNiPer Station, and its application for quantitative discrimination of Dehalococcoides species, which perform anaerobic dechlorination of chloroethenes. MagSNiPer Station is equipped with a thermal cycler, a tip stand, a microtitre-plate automated stacker, an eight-channel tip dispenser, a magnetic separation unit for Magtration technology, and a chemiluminescence detector. It can automatically perform all processes required for SNP genotyping by MagSNiPer. A primer was designed for discriminating single nucleotide difference between 16 S rRNA genes of Dehalococcoides ethenogenes and Dehalococcoides BAV1. Chemiluminescence intensities for the 16 S rRNA genes obtained by MagSNiPer were proportional to their quantity. MagSNiPer analysis of 16 S rRNA genes amplified on the DNA purified from groundwater gave a ratio of these two 16 S rRNA genes similar to that obtained by cloning and sequencing. MagSNiPer is much easier, more rapid and more cost-effective than conventional sequencing. Compared with denaturing gradient-gel electrophoresis, MagSNiPer has the advantage of being quantitative. Therefore, by applying MagSNiPer at several sites where single base differences exist among Dehalococcoides species, it is possible to analyse Dehalococcoides consortia with ease, yielding useful information on anaerobic bioremediation of chloroethenes.

Pretreatment of polyamide monofilament with hydrochloric acid improves sensitivity of three-dimensional microarray, Bio-Strand.

A single-nucleotide-polymorphism-typing method using a novel three-dimensional DNA microarray, Bio-Strand, is promising because it is rapid, inexpensive and easily automated. It has been developed with the intent to overcome the drawbacks of conventional DNA microarrays, which use flat surfaces and impermeable materials such as glass slides; Bio-Strand as a novel DNA microarray, with its permeability, has a significantly improved stability compared with conventional DNA microarrays that use impermeable materials. In this study, we have developed a simple method of pretreating a polyamide monofilament to increase its surface area and to make it permeable, which makes Bio-Strand more sensitive and stable, allowing it to be adapted for clinical diagnostic applications. The fluorescence signal obtained with a nylon 6 monofilament pretreated under optimal conditions (hydrolysis by 5 M HCl/ethanol followed by washing with 50% ethanol and 100% ethanol) was significantly stronger than that obtained with an untreated monofilament.

Development of an automation system for single nucleotide polymorphisms genotyping using bio-strand, a new three-dimensional microarray.

Previously, we developed a novel three-dimensional microarray system called Bio-Strand, which may be used in various applications including single nucleotide polymorphisms genotyping. In Bio-Strand, samples for detection are immobilized on a one-dimensional thread, which is wound around a cylinder-shaped core to form a three-dimensional thread-and-core structure. The thread-and-core structure is then inserted into a plastic pipette tip, where hybridization and detection are performed. In this study, we have developed an automation system, NIAGALA Bio-Station SDx, which enables automated hybridization and detection during the genotyping procedure using Bio-Strand. Using this system, we have performed the single nucleotide polymorphism (SNP) genotyping of CYP2C, one of the important human cytochrome P450 genes and the results were completely consistent with the genotyping results determined by the TaqMan method.

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence.

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.

Development of an integrated automation system with a magnetic bead-mediated nucleic acid purification device for genetic analysis and gene manipulation.

We have developed an integrated automation system for genetic analysis and gene manipulation. The system, SX-8G Plus, is equipped with an 8-nozzle dispensing unit, a thermal cycler, a cooled reagent reservoir, four tip storage racks, four microplate platforms, buffer reservoirs, an agarose gel electrophoresis unit, a power supply, a pump for exchanging electrophoresis buffer, and a CCD camera. Automation of nucleic acid extraction and purification, the most difficult step in automating genetic analysis and gene manipulation, was realized using magnetic beads with Magtration Technology, which we have previously developed for automating the handling of paramagnetic beads. Using this system, we could perform the automated separation and purification of DNA fragments by agarose gel electrophoresis starting from sample loading. The system would enable the automation of almost all procedures in genetic analysis and gene manipulation.