Kunio Harada

Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed‐side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel‐based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.

A new single nucleotide polymorphisms typing method and device by bioluminometric assay coupled with a photodiode array

Easy and inexpensive single nucleotide polymorphisms (SNPs) typing systems are required for the practice of genetic testing as well as genetic medicine. Most of the SNPs typing systems use laser-induced fluorescence detection coupled with fluorophore tagging on DNA, which are expensive. A new simple and inexpensive SNPs typing system is presented. It uses a bioluminometric assay coupled with modified primer extension reactions and an inexpensive photodiode array for the luminometric detection. The reagents consumed in the assay are also inexpensive. Although the system is very small, simple and inexpensive, it gives enough sensitivity for detecting target DNAs as small as 10 fmol, which is good enough for SNPs typing.

An automated liquid handling system for polymerase chain reaction sample preparation.

An automated liquid handling system for dispensing liquid samples of sub-microliter volume has been developed. The system has eight nozzles composed of glass capillaries connected to syringe-style pumps. The distance between the nozzles can be changed from 4.5 to 9 mm, which corresponds to the distance between the wells in 96- and 384-well microplates, respectively. The eight nozzles of the system can aspirate and dispense liquid samples in both 96- and 384-well microplates. Sub-microliter volumes of reagents and samples were transferred between 96- and 384-well microplates using the system. This system was successfully used for PCR sample preparation.

Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion

To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

Abstract 1574: KRAS genotyping by digital PCR combining melting curve analysis

Background ctDNA is a remarkable liquid biopsy for cancer diagnosis. Highly sensitive quantification method is required to measure a tiny amount of ctDNA. Digital PCR has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR does. However, the digital PCR has large fluctuation in the fluorescence intensity of the droplets or chambers resulting in lower accuracy. Main cause is probably due to the insufficient PCR in the small partitions. In this study, we have proposed a new measurement method combined a digital PCR with melting curve analysis using molecular beacons to solve above mentioned problems, and applied it to KRAS genotyping. Methods Molecular beacons, which have hydrophobic moiety in the stem, were designed for detecting KRAS mutation. The digital PCR with combination of asymmetric PCR was performed using the molecular beacons in the droplets. After the PCR, the fluorescence of the droplets was observed with a microscope while changing the temperature. A melting curve was prepared from the change in fluorescence intensity of the droplet, and the melting temperature (Tm) was calculated from the differential melting curve. Results The melting curve analysis for the KRAS mutation was performed in the droplets where the asymmetric PCR was performed using molecular beacons with hydrophobic stem, which improved signal-to-noise ratio of melting curves. The use of molecular beacons with hydrophobic stem can keep a background fluorescence at a constant value even at high temperature. The change in fluorescence intensity of PCR solution using molecular beacons with hydrophobic stem during the measurement was one-tenth of that using molecular beacon without hydrophobic stem. The asymmetric PCR enabled us to increase the amount of the PCR products hybridized with molecular beacons, resulting in the increase in the fluorescence intensity. The KRAS genotyping of wild-type (WT) and G12D mutant was conducted by the melting curve analysis with a combination of the asymmetric PCR with molecular beacons. The results showed that the peaks of the distributions of the Tm values of DNA in the droplets were 77.9°C for WT and 74.0°C for G12D mutant, which indicates that the WT and the mutant could be successfully discriminated by the proposed method. Conclusion We have proposed a new measurement method combining digital PCR with asymmetric PCR using molecular beacons and melting curve analysis. The genotyping of KRAS mutation was successfully performed by the proposed method. We are planning to prove the concept of this method for the clinical specimens in the future. Citation Format: Junko Tanaka, Yuzuru Shimazaki, Tatsuo Nakagawa, Akiko Shiratori, Masao Kamahori, Takahide Yokoi, Kunio Harada, Yoshinobu Kohara. KRAS genotyping by digital PCR combining melting curve analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1574.