Non-hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single-stranded DNA.

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label-free, and economical non-hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single-stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single-base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84-mer, in which guanine to adenine single-base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.

Separation of single-base sequential isomers of single-stranded DNA by capillary electrophoresis and its application in the discrimination of single-base DNA mutations.

Separation of single-base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single-base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single-base sequential isomers of 12-mer single-stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68-mer gene fragment and its single-base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single-base gene mutations.

Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry.

In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0-40.0, 0.10-1.60, and 0.13-2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were - 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.

Separation of oligonucleotides with single-base mutation by capillary electrophoresis using specific interaction of metal ion with nucleotide.

A unique tactic for the separation of single-base sequential isomers of oligomeric single-stranded DNA by a CE separation system employing the specific interaction of metal ion with nucleotide was demonstrated, enabling the separation of the mixture of a 12-mer oligonucleotide and its single-base mutants, as well as their positional isomers.

Color Change of Sudan III against Concentrated Sulfuric Acid in Acetonitrile and Quantification for a Small Amount of Concentrated Sulfuric Acid.

The color-changing phenomenon of hydrophobic bisazo dye, Sudan III in an acetonitrile solution against the addition of concentrated sulfuric acid has been discovered and the chromic properties investigated. Based on observations, a novel quantification method of concentrated sulfuric acid has been developed. Sudan III changes its color from orange to blue against a small volume of sulfuric acid, and the acetonitrile solution of Sudan III is the most suitable for observing the color-change phenomenon. (1)H-NMR and UV-Vis spectroscopic studies showed that the color-change mechanism of Sudan III against sulfuric acid is due to the protonation of the dye by sulfuric acid. This phenomenon is applicable to the quantification of concentrated sulfuric acid by introducing the Hammett acidity function. The proposed method requires only a small amount of the sample, 0.04 mL, and enables rapid quantification.

Development of a novel optical sensing method for acid solution using Oil Red O supported by florisil.

A novel field sensing method for concentrated acid solutions was developed. The sensor is composed of a dye, Oil Red O, and florisil as a support for the dye. When the dye is supported on the florisil surface, its color change properties against the acid solution drastically changes compared to in solution, and the sensor is applicable to sensing for acids of relatively low concentration. The significant phenomenon could not be observed when silica gel was used as a support, suggesting that the florisil plays an important role in the color changing phenomenon. The amount of dye absorbed on the florisil surface is related to the color change properties.

Pooled model-based approach to compare the pharmacokinetics of entecavir between Japanese and non-Japanese chronic hepatitis B patients.

This study evaluated the population pharmacokinetics (PK) of entecavir in Japanese patients with chronic hepatitis B infection enrolled in 2 Japanese phase IIb clinical trials and compared them to non-Japanese patients enrolled in global phase II trials. The objectives were to identify significant and clinically meaningful covariate effects on entecavir population pharmacokinetic parameters and assess whether differences exist between Japanese and non-Japanese patients. A total of 843 observations were obtained from 142 patients who received once daily administration of entecavir at 0.1, 0.5, and 1.0 mg doses in the 2 Japanese studies. Consistent with findings in non-Japanese patients, creatinine clearance estimated with ideal body weight (ICrCL) was found to be statistically significant for clearance in a 2-compartment model. Also, the entecavir dose was identified as a covariate on intercompartmental clearance. Age, gender, and hepatic function were not identified as covariate for clearance. The estimated population average of oral clearance in a typical patient with a reference ICrCL value of 100 mL/min was 26.4 L/h (interindividual variability: 19.4%). This model-based analysis indicates that the PK of entecavir are similar in Japanese and non-Japanese chronic hepatitis B patients.