Jun Komatsu

Single-molecule PCR using water-in-oil emulsion.

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.

One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface.

Abstract We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. γ DNA molecules were ligated with 3′-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified γ DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified γ DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.

Stretching of long DNA molecules in the microvortex induced by laser and ac electric field

A microvortex is generated around an infrared laser focus where an intense ac electric field is applied. The authors used this optoelectrostatic microvortex for stretching individual long DNAs. When λ-or T4-phage DNA molecules were introduced into the optoelectrostatic microvortex, they were stretched around the laser focus. In addition, especially for longer T4 DNA molecules, it was possible to keep it in stretching form for more than 30s. Using this method, length of DNA molecules can be measured without fixing to a substrate. This method can be applied to DNA molecules longer than about 10μm.

Activation of restriction enzyme by electrochemically released magnesium ion

Observation and cutting of DNA molecules at intended positions permit several new experimental methods that are completely different from conventional molecular biology methods; therefore several cutting methods have been proposed and studied. In this paper, a new cutting method for a DNA molecule by localizing the activity of a restriction enzyme is presented. Since most restriction enzymes require magnesium ions for their activation, local restriction enzyme activity can be controlled by the local concentration of magnesium ions. Applying a direct current (dc) voltage to a needle electrode of metallic magnesium made it possible to control the local magnesium ion concentration at the tip of the needle. The restriction enzyme was activated only when magnesium ions were electrochemically supplied.

Ice-water interface migration by temperature controlling for stretching of DNA molecules

Abstract This report shows a new DNA stretching method using migration of an ice-water interface. DNA molecules were stretched accompanying the migration of the solid-liquid interface and immobilized in frozen area. This simple method needs no chemical modification to keep DNA in the stretched form. For full stretching of DNA molecules, one terminus of the DNA molecules were anchored on silanized substrate. The anchored DNA molecules were stretched by freezing the DNA solution. The stretched DNA molecules were observed after sublimation of the frozen solution keeping its stretched form on silanized surface which had no attractive interaction with DNA molecules except for the SH-modi- fied terminus in solution. An infrared (IR) laser beam was introduced to a frozen DNA solution through an objective lens for local area melting of the solution. Scanning of the laser irradiation caused stretching and enclosing of DNA molecules in the frozen area followed by migration of the solid-liquid interface.

Attachment of heavy metal to DNA molecules for rapid analysis

Labeling of DNA has been studied using base-specific Rhodium (II) acetate [Rh/sub 2/(O/sub 2/CCH/sub 3/)/sub 4/] (Rh1) compound. First, DNA and Rh1 were mixed and the binding reaction was checked by electrophoresis of the complex. The result showed that some of DNA molecules were labeled by the Rh1. AFM observation also showed some overlapped DNA molecules. It suggests that DNA molecules could be cross-linked by Rh1 on the overlapped points.

Control of restriction enzyme activity by local concentration of magnesium ion

Observation and cutting of DNA molecules at arbitrary positions permit several new experimental methods that are completely different from conventional methods of molecular biology, therefore several cutting methods have been proposed and studied. In this report, a new cutting method for a DNA molecule by localizing the activity of restriction enzyme is presented. Since most restriction enzyme requires magnesium ion for their activation, the local restriction enzyme activity can be controlled by the local concentration of magnesium ions. The local concentration of magnesium ions can be controlled electrochemically by applying a DC voltage to a needle electrode of magnesium metal. The restriction enzyme can be activated only when magnesium ion is electrochemically supplied.