DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3′ end of the immobilized DNA primers on the S-Bio® by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3′-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

The rapid identification of bacteria in many kinds of samples, i.e., clinical, food, water, and material, is important from a hygienic standpoint. In this paper, we describe the development of a convenient bacterial identification system using bacterial cells by a plastic DNA array. The small plastic base, which was cut from an S-BIO PrimeSurface plastic base developed for the covalent immobilization of aminomodified DNA, was used as a substitute for a glass base. The species-specific primers were immobilized on the small plastic base and the spots specific to each bacterial species were observed after 2 types of thermal cycles in a single PCR tube using bacterial culture broth as a sample. The results obtained using the culture broth were the same as those obtained with total DNA extracted from bacterial cells. The detection limits of Staphlococcus aureus (S. aureus) ATCC25923 and Escherichia coli (E. coli) ATCC25922 were 8.7 × 10 3 cells/µl and 2.1 × 10 2 cells/µl, respectively. This system is useful and convenient for the identification of bacteria in many types of samples. Moreover, further improvements in conditions, such as the ingredients in the reaction mixture, thermal cycles, and the steps of visualization would result in a more efficient system of identification.

It was found that Serratia marcescens 43, Serratia proteamaculans 48 and Serratia sp. 45, all of which were clinically isolated, produced a new type of aminoglycoside acetyltransferase which acetylated amikacin at the 6'-amino group. 1-N-[(S)-3-Amino-2-hydroxypropionyl]gentamicin B (HAPA-B, SCH 21420) and gentamicin C2 were hardly inactivated by the enzymes and had effective antimicrobial activities against these strains both in vitro and in vivo. This kind of aminoglycoside acetyltransferase should be classified into a new group other than previously reported AAC(6') enzymes.