Eisai R&d Management Co., Ltd.

Sanko Junyaku Co., Ltd.

Palma Bee'z Research Institute Co., Ltd.

Eisai & Managment Co., Ltd.

Mitsugu Usui

Chikako Hakii

Mari Mitsuka

Toshihiko Fujikawa

Tatsuo Ichihara

Akiji Aoki

Motohito Kaneshima

Mari Yamaguchi

Provided are a method of detecting a target substance, by which the detection sensitivity in the PALSAR method can be improved and multiple genes can be simultaneously detected, and a kit for detection. The method is a method of detecting a target substance by forming a signal probe polymer with the use of multiple dimer probes or dimer-forming probes and an assist probe, in which each dimer probe is made up of two kinds of dimer-forming probes including a 5'-side region, a central region and a 3&#x2032;-side region (the central regions of the two kinds of dimer-forming probes are complementary to each other and the 3&#x2032;-side regions and the 5&#x2032;-side regions thereof are not complementary to each other), the multiple dimer probes are constituted such that a dimer probe polymer is formed via a self-assembly reaction, and the assist probe is designed so as to have a structure having a region complementary to the 5&#x2032;-side region of one dimer-forming probe in one dimer probe, a region complementary to the 3&#x2032;-side region of the other dimer-forming probe, and a target region capable of binding to the target substance.

Method of detecting target substance

Provided are a method of forming a signal probe-polymer which makes it possible to form a polymer efficiently and quantitatively, a signal probe-polymer formed by the method, oligonucleotide probes for use in the method, and a method of detecting target analyte having high sensitivity and excellent quantitative capability. The method of forming a signal probe-polymer comprises reacting a plurality of pairs of oligonucleotide probes with each other to form a polymer, a first probe of the pair of oligonucleotide probes comprising three nucleic acid regions of X, Y, and Z regions, located in the stated order from the 5'-terminal and a second probe comprising three nucleic acid regions of X&#x2032;, Y&#x2032;, and Z&#x2032; regions, located in the stated order from the 5&#x2032;-terminal, wherein each region of the oligonucleotide probes has a length of from 13 to 15 bases.

Method of forming signal probe-polymer

To solve a problem occurring in the PALSAR method that a polymer would be formed in the state of unbound to a captured test gene and thus affect the quantitative characteristics as a nonspecific signal, it is intended to develop a technique whereby the polymer formation is controlled in the step of forming an assembly (polymer) of probes so that the polymer is formed exclusively on a test gene to thereby improve the sensitivity and quantitative characteristics. It is found that the polymer can be quantitatively formed and a nonspecific reaction can be inhibited by, in the step of forming a polymer by reacting plural kinds of probes having abilities to complementarily bind to each other, not adding or reacting these probes at once but starting with the reaction of a first probe in one group, and then reacting the second probe in the other group followed by the reactions of probes one by one (i.e., the first probe, the second probe, and so on).

Provided are a method of detecting target genes capable of increasing sensitivity in a Palsar method and of simultaneously detecting multiple genes, an assist probe to be used in the above method, and a method of forming a signal probe polymer by using the assist probe. The method of detecting target genes includes: forming a signal probe polymer by using a first probe having a nucleic acid region X, a nucleic acid region Y and a nucleic acid region Z in the stated order from the 5' end, a second probe having a nucleic acid region X&#x2032;, a nucleic acid region Y&#x2032; and a nucleic acid region Z&#x2032; in the stated order from the 5&#x2032; end, and an assist probe having a plurality of the same nucleic acid regions as in the first probe and a target region capable of hybridizing with a target gene. In this method, the assist probes are designed so as to have a structure including the nucleic acid regions X, Y and X, and the target region in the stated order from the 5&#x2032; end or a structure including the target region, and the nucleic acid regions Z, Y and Z in the stated order from the 5&#x2032; end.

Assist probe and method of using the same

A hybridization method is provided in which an efficient hybridization reaction can be carried out. Further, there are provided, using this hybridization method, a method for detecting a target gene with high sensitivity and a signal amplifying method for markedly improving the detection sensitivity of the target gene. There is provided a hybridization method comprising the use of oligonucleotides in a reaction solution, the method comprising forming partially a reaction temperature region in the reaction solution and performing a hybridization reaction in the reaction temperature region.

Hybridization method

There is provided a signal amplification method for detecting a mutated gene, which can increase the detection sensitivity of mutated genes on a DNA chip according to the PALSAR method, can establish efficient signal amplification and can establish simple detection by contriving design of oligonucleotide probes for use in the PALSAR method. The signal amplification method comprises a ligation reaction with a DNA ligase and a self-assembly reaction which forms a double-stranded self-assembly substance having a regular higher-order structure of oligonucleotides, wherein the detection sensitivity of the mutated gene on a DNA chip is improved.

Signal amplification method for detecting mutated gene

There is provided a method for forming a self-assembly substance using oligonucleotides without using special instruments or complicated procedures, an self-assembly substance formed by the method for forming the self-assembly substance, and a method for detecting an amplified target gene at a low cost and in a simple way by making use of the method for forming the self-assembly substance. In the method for forming the self-assembly substance using a self-assembly reaction of oligonucleotides, the oligonucleotides comprise oligonucleotides synthesized by a gene amplification reaction. The oligonucleotides synthesized by the gene amplification reaction are detected by forming a self-assembly substance by the use of the method for forming the self-assembly substance of oligonucleotides, and by detecting the formed self-assembly substance.

Method for forming self-assembly substance using oligonucleotide synthesized by gene amplification reaction, self-assembly substance and method for detecting gene

The present invention provides a novel method for forming a self-assembly substance of probes making it possible to shorten a reaction time required for formation of a self-assembly substance and also to increase regions of probes available for detection of a target gene by previously preparing a plurality of dimer-probes and increasing the types. This method comprises the steps of: providing plural groups, wherein each group includes a pair of dimer forming probes containing a pair of oligonucleotides, each oligonucleotide having three regions of a 3' side region, a mid-region and a 5&#x2032; side region, in which the mid-regions of the oligonucleotides have base sequences complementary to each other, and the 3&#x2032; side regions and the 5&#x2032; side regions of the oligonucleotides have base sequences not complementary to each other; hybridizing a plurality of pairs of the dimer forming probes of the plural groups; and forming a double-stranded self-assembly substance by self-assembly of the oligonucleotides.

Methods of constructing self-assembly of probes and method of detecting the same

The present invention provides a method for measuring a target gene under isothermal conditions without using enzyme. A pair of probes each having n (n&#x2267;3) base sequence regions complementary to each other are hybridized alternately to form a double-stranded probe-polymer. A base pair at branched sites of each complementary base sequence region is designed to be a G (guanine)-C (cytosine) bond, whereby a stable double-stranded probe-polymer is formed. One of complementary portions in one probe is constituted to have a base sequence complementary to a part of a target gene, whereby a target gene-probe-polymer complex is formed and the target gene is measured.

Probe for constructing probe-polymer method of constructing probe-polymer and utilization thereof

Coprecipitant and method for extracting nucleic acids

A gene amplifying method for efficiently amplifying a gene without using enzyme or branched DNA. A plurality of pairs of probes each composed of three or more portions complementary to such portions in the other probe are used, and hybridized such that they cross in alternation to form a double-stranded polymer.

Growing community of inventors

Yokohama, Japan

Mitsugu Usui