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S Tamatsukuri

Publications and source records attributed to S Tamatsukuri.

7 recordsLinked to original sources
PubMed1992

A new method for constructing NotI linking and boundary libraries using a restriction trapper.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.

Base Sequence↗
PubMed1992

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.

Alkaline Phosphatase↗
PubMed1991

Detection of a heat-labile enterotoxin gene in enterotoxigenic Escherichia coli by densitometric evaluation using highly specific enzyme-linked oligonucleotide probes.

Two alkaline phosphatase-conjugated 24-mer oligonucleotide probes were developed to detect the heat-labile enterotoxin gene in enterotoxigenic Escherichia coli. Probes were antisense codon sequences, which are transcribed into mRNA, of the heat-labile enterotoxin gene of enterotoxigenic Escherichia coli of human origin. Using dot-blot hybridization, probes were tested with 100 clinical isolates and evaluated by a reflectance-type densitometer. Results agreed very well with those of an immunological test, the Biken test, and a 32P-labelled recombinant DNA probe. The oligonucleotide probes did not react with nucleic acids prepared from other diarrhoeagenic bacterial pathogens. Thus, the alkaline phosphatase-conjugated oligonucleotide probes seem to be highly sensitive and specific for detection of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli. Moreover, the results indicate a potential usefulness for densitometric evaluation of DNA hybridization.

Alkaline Phosphatase↗
PubMed1987

Solid phase synthesis of oligoribonucleotides using the o-nitrobenzyl group for 2'-hydroxyl protection and H-phosphonate chemistry.

Oligoribonucleotides with chain length of 7, 11, 15, 17, 24 and 34 were synthesized on long chain alkylamine controlled pore glass beads (LCA-CPG) using o-nitrobenzyl protection of 2'-hydroxyls via a H-phosphonate approach either manually or by using an automatic synthesizer. The oligoribonucleotides were obtained in yields of 0.6 0.6-20%, based on initial nucleoside bound to the LCA-CPG support.

Automation↗
PubMed1986

Solid phase synthesis of oligoribonucleotides using o-nitrobenzyl protection of 2'-hydroxyl via a phosphite triester approach.

The hepta and undecaribonucleotide were synthesized on a controlled pore glass beads using o-nitrobenzyl protection of 2'-hydroxyls via a phosphite approach. By using 5-p-nitrophenyltetrazole for the activation of nucleoside-phosphoramidite, the condensation reaction was carried out very rapidly (2.5 min). The time required for one cycle was only 16 min. The hepta-(UACUAAC) and undecaribonucleotides (GUAUGUUAAUA) were obtained in yields of 28 and 17% respectively from the original resin.

Methods↗
PubMed1986

Synthesis of 5'-phosphorylated oligonucleotides on a polymer support.

Highly lipophilic trityloxyethylamine (TEAm) or trityloxyethylaniline (TEAn) were prepared and used for the protection of the 5'-terminal phosphate. We synthesized 5'-phosphorylated oligodeoxyribonucleotides by the solid phase synthesis. TEAm or TEAn groups enabled us to isolate the desired oligonucleotide easily by the reversed phase column due to their high lipophilicity and could be removed by the aq. AcOH treatment.

Base Sequence↗