Toyo Boseki Kabushiki Kaisha

Yutaka Takarada

Yoshihisa Kawamura

Katsuya Daimon

Shigeru Komai

Shuji Shibata

Hiroaki Inoue

Atsushi Sogabe

Toshihiro Kuroita

Yoshihiro Soya

Motohiro Kondo

Toshiya Aono

Masaya Segawa

Naoki Tanaka

A method for identifying a nucleotide polymorphism, the method comprising hybridizing a labeled oligonucleotide for a wild type or a mutant type to a nucleic acid containing a specific nucleotide polymorphic site in a sample; allowing a nucleic acid-specific label to act thereon; detecting an interaction between the label of the oligonucleotide and the nucleic acid-specific label; and identifying a nucleotide polymorphism, wherein the identification utilizes a difference due to the nucleotide polymorphism in conditions under which the hybridized oligonucleotide is separated into a single strand.

Method for identifying nucleotide polymorphism

It is intended to provide a method of conveniently screening a novel protein or a novel partial sequence protein having a blocking ability based on amino acid sequence data; and a protein achieving an improved blocking efficiency that can be expressed on a large scale in. A method of screening a novel protein or a novel partial sequence protein having a blocking ability based on amino acid sequence data; a protein characterized by achieving an improved blocking efficiency owing to an amino acid sequence modification; and a method of utilizing the protein.

Protein achieving improved blocking efficiency

The present invention provides a method of extracting and isolating nucleic acids from a material containing nucleic acids using a nucleic acid-binding particulate carrier. More specifically, the present invention provides a nucleic acid extraction method using a particulate carrier having a particle diameter of 0.5 to 15.0 &mgr;m, a pore diameter of 50 to 500 nm and a pore volume of 200 to 5000 mm /g. According to the method of the invention, nucleic acids can be efficiently extracted from a biological material, in particular a material containing a large amount of contaminants, such as a clinical sample.

Method of extracting nucleic acids using particulate carrier

In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from,which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.

Method for amplifying and detecting of target nucleic acid sequence using thermostable enzyme

This invention provides an apparatus for extracting nucleic acids from nucleic acid-containing samples. The nucleic acid extraction apparatus of the invention comprises (1) a group of extraction vessels each comprising a reactor tube in which a sample, a reagent solution, and a magnetic carrier are admixed and reacted, a drain cup for pooling an unwanted component solution, and a nucleic acid recovery tube all as secured to a support, (2) a distribution means for introducing a solution into each of the extraction vessels, (3) a stirring means for mixing the solution and magnetic carrier in the reactor tube, (4) a holding means for holding the magnetic carrier stationary within the vessel, (5) a discharging means for discharging the solution from the reactor tube while the magnetic carrier is held stationary, (6) a heating means for heating the solution and magnetic carrier in the reactor tube, and (7) a transfer means for serially transferring the vessels to the given positions.

Nucleic acid extraction apparatus

The present invention provide a process for sequence-specific nucleic acid amplification capable of improving the detection sensitivity and increasing the signal. In particular, the present invention provides a reagent for nucleic acid amplification containing at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts, specifically a reagent for nucleic acid amplification comprising, in addition to the at least one compound, a forward primer having a DNA sequence homologous to a sequence of a target RNA; a reverse primer having a DNA sequence complementary to a sequence of the target RNA and having a promoter for RNA polymerase attached to its 5&prime; end; ribonucleotides; deoxyribonucleotides; a reverse transcriptase or RNA-directed DNA polymerase; a RNase H; a DNA polymerase or a reverse transcriptase having DNA-directed DNA polymerase activity; and a RNA polymerase. The present invention also provides a process for nucleic acid amplification characterized by carrying out a nucleic acid amplification reaction in the presence of at least one member selected from the group consisting of EDTA, NTA, UDA, CyDTA, DTPA, GEDTA, TTHA and their salts.

Reagent for nucleic acid amplification and process for nucleic acid amplification

This invention provides an apparatus for extracting nucleic acids from nucleic acid-containing samples, particularly biological samples, and more particularly to a nucleic acid extraction apparatus well suited for the nucleic acid extraction method utilizing a nucleic acid-binding magnetic carrier. The nucleic acid extraction apparatus of the invention comprises (1) a group of extraction vessels each comprising a reactor tube in which a sample, a reagent solution, and a magnetic carrier are admixed and reacted, a drain cup for pooling an unwanted component solution, and a nucleic acid recovery tube all as secured to a support, (2) a distribution means for introducing a solution into each of the extraction vessels, (3) a stirring means for mixing the solution and magnetic carrier in the reactor tube, (4) a holding means for holding the magnetic carrier stationary within the vessel, (5) a discharging means for discharging the solution from the reactor tube while the magnetic carrier is held stationary, (6) a heating means for heating the solution and magnetic carrier in the reactor tube, and (7) a transfer means for serially transferring the vessels to the given positions where the distribution means, stirring means, holding means, discharging means and heating means are respectively disposed.

Nucleic acid extraction method

In a method for amplifying the specific nucleic acid sequence, a highly specific amplification which has low possibility of non-specific hybridization can be carried out. Highly stable reagents, the activities of which do not decrease in case of supply and storage, are also provided. Thermostable enzymes are used as RNA dependent DNA polymerase, DNA dependent DNA polymerase, DNA dependent RNA polymerase and ribonuclease H, which are required for the amplification system based on replicated RNA. Especially, it is preferable that a thermostable enzyme derived from Thermus thermophilus which has RNA dependent DNA polymerase activity, DNA dependent DNA polymerase activity and ribonuclease H activity, and thermostable DNA dependent RNA polymerase are used together. By this method, inactivation of enzymes are prevented by using thermostable enzymes, and the amplification can be carried out without sequential addition of enzymes.

Method for amplifying and detecting of target nucleic acid sequence

An oligonucleotide is provided, which has at least a base sequence A, which is 10-30 nucleotides in length and homologous to a part of a specific nucleic acid sequence to be amplified, and a base sequence B, which is 10-30 nucleotides in length and complementary to a sequence 3' to the part of the specific nucleic acid sequence to be amplified. Sequences A and B are separated by a spacer region of 0-20 nucleotides. The oligonucleotide is used in an amplification method, wherein the oligonucleotide is mixed with a sample containing a specific nucleic acid sequence to be amplified and allowed to anneal to the specific nucleic acid sequence in the sample. The annealed oligonucleotide acts as a primer in an elongation reaction, whereas any nonannealed oligonucleotide is decomposed, except for at least part of the base sequence A. The elongation product is then denatured to a single strand for use as a template to which the remaining oligonucleotide anneals to form a primer for subsequent elongation. Also provided is a detection method, which allows for detection of a specific nucleic acid sequence in a sample, as well as reagent kits for use in the amplification and detection methods.

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Otsu, Japan

Yutaka Takarada