Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.

We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

Instrumentation of a PLC-regulated temperature cycler with a PID control unit and its use for miniaturized PCR systems with reduced volumes of aqueous sample droplets isolated in oil phase in a microwell.

We have developed a temperature cycler for polymerase chain reaction (PCR) in a microwell fabricated on a polymer/glass chip. The entire system consisted of three subsystems, which included (1) a thermal conditioner, (2) a proportional-integral-derivative (PID) control signal conditioner and (3) a data acquisition subsystem. The subsystems were regulated coordinately by a ladder logic program written for the programmable logic control (PLC) so that an actual sample temperature could be timed, changed and maintained according to the programmed temperature cycles. The present temperature control system showed high accuracy, stability and minimum overshoot with reduced heating and cooling transition rates. Applicability of the temperature controller to the miniaturized PCR system with reduced volumes of aqueous sample droplets isolated in an oil phase was confirmed by successful amplifications of a target DNA sequence in the microwell.

Compact polytetrafluoroethylene assembly-type capillary electrophoresis with chemiluminescence detection.

We have developed a compact polytetrafluoroethylene (PTFE) assembly-type capillary electrophoresis with chemiluminescence (CL) detection system. Luminol-microperoxidase-hydrogen peroxide chemiluminescence reaction was adopted. The device is rectangular in shape (60 mm x 40 mm x 30 mm) and includes three reservoirs (sample, migration buffer, and detection reservoirs) with electrodes. The detection reservoir includes an optical fiber to transport light at the capillary tip to a photomultiplier tube. Isoluminol isothiocyanate (ILITC) was analyzed as a model using this device with fused-silica or polytetrafluoroethylene capillary tubes 10 cm in length. We also used the sample reservoir as a reactor for an immune reaction between anti-human serum albumin immobilized on glass beads and isoluminol isothiocyanate-labeled human serum albumin. The present polytetrafluoroethylene assembly with the capillary tube was useful as a palm-sized analysis device for separation and detection, as well as a reactor.

Molecular recognition of mono- and disaccharides through interaction with p-iodophenylboronic acid in capillary electrophoresis with a chemiluminescence detection system.

Molecular recognition of mono- and disaccharides was performed making use of the interaction between their diol groups and p-iodophenylboronic acid in capillary electrophoresis (CE) with a chemiluminescence (CL) detection system. p-Iodophenylboronic acid acted as an enhancer for luminol-horseradish peroxidase-hydrogen peroxide CL reaction. p-Iodophenylboronic acid was injected as a sample into the present system to give a CL peak on the electropherogram. The CL intensities were examined using running buffers including mono- and disaccharides. The CL intensities with 1-methyl-D-glucoside, D-saccharose, D-maltose, D-glucose, and D-fructose decreased in this order. The decrease in CL intensity was based on the formation by p-iodophenylboronic acid of cyclic esters with mono- and disaccharides, particularly with those including cis-diol groups. That is, the decrease in CL intensity affected the specific complexation between p-iodophenylboronic acids and saccharides, leading to the molecular recognition of saccharides. We also report separation of a mixture of p-iodophenol and p-iodophenylboronic acid as well as estimation of the apparent binding constant between p-iodophenylboronic acid and saccharides taking advantage of their molecular recognition behavior.

Chemiluminescence from singlet oxygen under laminar flow condition in a micro-channel.

Singlet oxygen was generated by reaction of sodium hypochlorite and hydrogen peroxide in a micro-channel. The two reagent solutions were delivered into the micro-channel by syringe pumps, providing a laminar flow. Such a laminar flow forms a liquid-liquid interface instantly in a micro-channel, and then the interface collapses gradually through molecular diffusion with the residence times. The chemiluminescence from the singlet oxygen was emitted in the course of the collapse of the interface under laminar flow condition. The chemiluminescence intensity was observed continuously and stably in the micro-channel as long as the reagents were fed into the channel. We examined the features of the chemiluminescence emitted in the micro-channel by changing the flow rates of reagents and the detection points in the micro-channel. The data obtained were considered along with the residence times and diffusion lengths. We also examined the effects of antioxidants, such as sodium azide, histidine, nitroblue tetrazolium, and 2-propanol on the chemiluminescence intensity.

[A case of emergency surgery in a patient with bronchial asthma under continuous spinal anesthesia].

A 78-year-old male, observed for bronchial asthma, underwent two emergency operations within eight days. The first operation was performed under general anesthesia with tracheal intubation. Anesthesia was maintained by sevoflurane-oxygen and continuous infusion of propofol in combination with epidural injection of lidocaine. During the operation, respiratory sound was almost clear. But wheezing occurred as he awoke after discontinuation of the anesthetics. He needed ventilatory support for three days for status asthmatics. The second operation was performed under continuous spinal anesthesia using hypobaric tetracaine and hyperbaric bupivacaine. No ventilatory support was necessary after the operation and he was discharged uneventfully.