Sanko Junyaku Co., Ltd.

Eisai R&d Management Co., Ltd.

Eisai & Managment Co., Ltd.

Chikako Hakii

Mitsugu Usui

Mari Mitsuka

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

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 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.

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Yokohama, Japan

Chikako Hakii