Aisin Seiki Kabushiki Kaisha

Riken Corporation

Aisin Cosmos R&d Co., Ltd.

Kazusa Dna Research Institute

Kazusa Dna Research Institute Foundation

Yasushi Shigemori

Michio Oishi

Tsutomu Mikawa

Takehiko Shibata

Osamu Ohara

Jun Fujiwara

Yuichiro Nakaoki

Atsushi Hattori

An objective of the present invention is to establish a technique for making it possible to immobilize an enzyme on an electrically conductive base material in a uniformly, high density, and constantly aligned orientation, for the purpose of constructing an enzyme electrode having improved electrode performance. An electrode having enzyme crystals immobilized thereon, the electrode being provided with an electrically conductive base material that can be connected to an external circuit and enzyme crystals that serve as an electrode catalyst, wherein the enzyme crystals are immobilized on the electrically conducive base material; a method for producing an electrode having enzyme crystals immobilized thereon; and a biological fuel cell and a biosensor which are provided with an electrode having enzyme crystals immobilized thereon.

Electrode having enzyme crystals immobilized thereon, method for producing electrode having enzyme crystals immobilized thereon, and biological fuel cell and biosensor provided with electrode having enzyme crystals immobilized thereon

The invention establishes a technology that allows non-specific amplification to be inhibited during nucleic acid amplification in an isothermal amplification reaction, such that the amplification efficiency is increased. The invention is a extreme thermophile single-stranded DNA binding mutant protein, having an amino acid sequence that expresses a function that can contribute to increasing an amplification efficiency of a template nucleic acid in an isothermal amplification reaction system that uses a strand displacement polymerase, and having in its amino acid sequence a mutation site where a mutation involving at least one of deletion, substitution, addition, and insertion of one or more amino acids in amino acid sequence of extreme thermophile single-stranded DNA binding protein has occurred, and a method of use thereof.

Extreme thermophile single-stranded DNA binding mutant protein, and nucleic acid isothermal amplification method of use thereof

A heat-stable RecA mutant protein is obtained by mutation involving either deletion or substitution of at least one amino acid in an amino acid sequence composing an acid region at C-terminal end of a wild type heat-stable RecA protein, and has an improved ability, compared to the wild type heat-stable RecA protein, for contributing to an increase in an amplification specificity of a template nucleic acid in a nucleic acid amplification reaction.

Heat-stable recA mutant protein and a nucleic acid amplification method using the heat-stable recA mutant protein

An object of the present invention is to provide a nucleic acid amplification method for amplifying a desired nucleic acid while suppressing amplification of byproducts in a PCR reaction, a reagent kit used for nucleic acid amplification, a method of detecting single nucleotide polymorphism to detect single nucleotide polymorphism by utilizing that amplification of byproducts is suppressed in a PCR reaction, and a reagent kit used for detecting single nucleotide polymorphism. The method of amplifying nucleic acids by PCR is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from, and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.

Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism

The present invention is to provide a method which makes it possible to activate RecA, and maintain its biological function in all the PCR cycles. Also, the invention is to provide a nucleic acid amplification method, and a kit for amplifying a nucleic acid which makes it possible to suppress non-specific amplification more specifically and efficiently to amplify only the desired nucleic acid. A method of activating a RecA protein derived from an extremely thermophilic bacterium in a polymerase chain reaction carried out in the presence of the RecA protein derived from the extremely thermophilic bacterium, wherein the RecA protein is activated by carrying out the reaction with the addition of nucleotide 5'-triphosphate (provided that the nucleotide 5&#x2032;-triphosphate is neither deoxynucleotide 5&#x2032;-triphosphate nor nucleotide 5&#x2032;-O-3-thiotriphosphate).

Activation method of protein derived from extremely thermophilic bacterium in nucleic acid amplification reaction and use thereof

The present invention relates to a DNA ligation method. Specifically, a DNA complex comprising a three-stranded structure in each of the ends of double-stranded DNAs, one of which has single-stranded regions at the end, and the other of which does not have single-stranded regions at the end, is formed under the presence of a homologous recombinant protein. Moreover, according to needs, the DNA complex is introduced into host cells, and said cells are cultured.

Ligation of double-stranded DNAs

An objective of this invention is to provide a method for detecting DNA polymorphism that has high sensitivity and efficiency and does not need long DNA searching region. A homologous recombination protein RecA makes partial triple strand DNA from target double DNA and oligonucleotide probe complementary to the DNA. The triple strand DNA maintains stable triple strand DNA after RecA protein is removed. The present inventors found that the thermostability of triple strand DNA changes greatly when there is a mismatch between target DNA and oligonucleotide probe because of the existence of polymorphism in the target DNA. Utilizing this change of thermostability, efficient detection of polymorphism in labeled DNA is possible by examining whether oligonucleotide probe is released and the triple strand DNA is solved after heat treatment of triple strand DNA formed using homologous recombination protein.

Method for detecting DNA polymorphism applying triple strand DNA formation technique

The present invention provides methods of preparing a triple-stranded DNA molecule by forming a DNA and protein complex, which DNA has a linear double-stand molecule and a single-strand molecule where the linear single-stranded DNA is complementary to a 5&prime; end region of one strand in the linear double stranded DNA; and the protein component is a homogeneous recombinant protein and an exonuclease; and then the protein is removed from the complex; triple-stranded DNA; and methods of using the triple-stranded DNA to detect nucleic acid sequence.

Triple-stranded DNA, method of forming the same and southern hybridization

A method for specifically cleaving a double-stranded DNA. The method comprises forming a three-stranded portion comprising the double-stranded DNA portion including the position to be cleaved or its vicinity and an oligonucleotide and treating the three-stranded protion with a nuclease.

Method for specifically cleaving double-stranded DNA and kit for the method

A preparation method of labeled DNA and use thereof are disclosed. More specifically, the invention relates to a preparation method of labeled DNA by preparation of a complex triple-stranded DNA through an deoxyoligonucleotide complementary to the deoxyoligonucleotide sequence of the 3&prime; terminal sequence of a certain double-stranded DNA followed by replacement of the 3&prime; terminal region of the plus strand of said double-stranded DNA with at least one labeled dNTP, and also to said immobilization method of a DNA obtained from the preparation method of the labeled (or modified) DNAs onto a solid support. These subject matters are mainly useful for preparation of labeled DNA molecules.

Preparation of labeled DNA

A nucleic acid detecting method using a probe is provided which makes it possible to obtain correct information on a target double-stranded DNA dsDNA without damaging the target double-stranded DNA dsDNA. In the nucleic acid detecting method of the present invention, a nucleic acid is detected by subjecting the target double-stranded DNA dsDNA bound with a first single-stranded DNA dsDNA probe via recA protein to a treatment with a single-stranded DNA dsDNA specific nuclease, to thereby allow a second single-stranded DNA dsDNA probe consisting of a base sequence complementary to that of the first probe or a part thereof and labeled with a detectable marker to bind to the target DNA dsDNA.

Method for detecting nucleic acids through a triple-stranded DNA

This invention provides a hairpin-shaped nucleic acid probe which is capable of forming stable hybrid with target nucleic acid, and a method for detecting nucleic acid containing a sequence complementary to a part of sequence of the probe. After contacting RecA to a hairpin-shaped single strand probe for hybridization, this complex is contacted with a target nucleic acid to form a triplex recombination intermediate using RecA function. After ligating the probe and the target nucleic acid, the target nucleic acid is detected by the label on the probe.

Growing community of inventors

Kisarazu, Japan

Yasushi Shigemori