KRAS genotyping by digital PCR combined with melting curve analysis.

Digital PCR (dPCR) has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR. However, dPCR exhibits large fluctuations in the fluorescence intensity of the compartment, resulting in low accuracy. The main cause is most likely due to insufficient PCR. In this study, we proposed a new method that combines dPCR with melting curve analysis and applied that method to KRAS genotyping. Since the melting temperature (Tm) of the PCR product hardly depends on the amplification efficiency, genotyping accuracy is improved by using the Tm value. The results showed that the peaks of the distribution of the Tm values of DNA in the wells were 68.7, 66.3, and 62.6 °C for wild-type KRAS, the G12R mutant, and the G12D mutant, respectively, and the standard deviation of the Tm values was 0.2 °C for each genotype. This result indicates that the proposed method is capable of discriminating between the wild-type sequence and the two mutants. To the best of our knowledge, this is the first demonstration of the genotyping of single mutations by combining melting curve analysis and dPCR. The application of this approach could be useful for the quantification and genotyping of cancer-related genes in low-abundance samples.

Far-ultraviolet absorbance detection of sugars and peptides by high-performance liquid chromatography.

A far-ultraviolet (FUV)-absorbance detector with a transmission flow cell was developed and applied to detect absorbance of sugars and peptides by HPLC. The main inherent limitation of FUV-absorbance detection is the strong absorptions of solvents and atmospheric oxygen in the optical system as well as dissolved oxygen in the solvent. High absorptivity of the solvent and oxygen decreases transmission-light intensity in the flow cell and hinders the absorbance measurement. To solve the above drawbacks, the transmission-light intensity in the flow cell was increased by introducing a new optical system and a nitrogen-purging unit to remove the atmospheric oxygen. The optical system has a photodiode for detecting the reference light at a position of the minus-first-order diffracted light. In addition, acetonitrile and water were selected as usable solvents because of their low absorptivity in the FUV region. As a result of these implementations, the detectable wavelength of the FUV-absorbance detector (with a flow cell having an effective optical path length of 0.5mm) can be extended down to 175nm. Three sugars (glucose, fructose, and sucrose) were successfully detected with the FUV-absorbance detector. These detection results reveal that the absorption peak of sugar in liquid phase lies at around 178nm. The detection limit (S/N=3) in absorbance with a 0.5-mm flow cell at 180nm was 21µAU, which corresponds to 33, 60 and 60µM (198, 360, and 360pmol) for fructose, glucose, and sucrose, respectively. Also, the peptide Met-enkephalin could be detected with a high sensitivity at 190nm. The estimated detection limit (S/N=3) for Met-enkephalin is 29nM (0.29pmol), which is eight times lower than that at 220nm.

Enzyme immunoassay using a reusable extended-gate field-effect-transistor sensor with a ferrocenylalkanethiol-modified gold electrode.

A reusable extended-gate field-effect transistor (FET) sensor with an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode was developed for applying to enzyme immunoassay. It was found that the 11-FUT modified FET sensor detected a thiol compound 50 times or more repeatedly after a treatment with a 5% hydrogen peroxide solution. The gate-voltage shift of the FET sensor showed a fairly good linearity (R(2) = 0.998) within a range from 10(-2) to 10(-6) M on the concentration of 6-hydroxyl-1-hexanethiol, which is a thiol compound, at a Nernstian response of 58.5 mV/decade. The FET-based immunoassay was constructed by combining the 11-FUT modified-FET sensor with the enzyme-linked immunosorbent assay (ELISA), in which the enzyme chemistry of acetylcholinesterase (AChE) was used to generate a thiol compound. The 11-FUT modified FET sensor with an AC voltage at 1 MHz superimposed onto the reference electrode detected the AChE-catalyzed product corresponding to a serum concentration of interleukin 1beta from 10 to 5000 pg/mL. In addition, all measurements were successfully performed by using the same FET-sensor chip after a treatment with a 5% hydrogen peroxide solution.

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis.

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.