Tetsuo Yukimasa

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3′ end were carried out in order to develop allele‐specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so‐called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3′ end were used. In particular, 3′ terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3′ terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele‐specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal‐to‐noise ratio of at least 45.

Surface Modification of Si3N4-Coated Silicon Plate for Investigation of Living Cells

We report a simple new method to improve the cell adhesion ability on the Si3N4 surface. Light-addressable potentiometric sensors (LAPS) have become a powerful tool for studying the biological action of cells. The Si3N4 surface of a LAPS structure was coated with poly-L-ornithine and laminin to improve its cell adhesion ability. The thickness of the coated layer was very thin, about 4 nm, as determined by atomic force microscopy measurement and thus this treatment surface is suitable for biological sensing applications such as the detection of cell activities.