Hitachi, Ltd.

Hitachi Electronics Engineering Co., Ltd.

Hitachi Chemical Company, Ltd.

Hitachi Software Engineering Company, Ltd.

Hitachi Chemical Research Center, Inc.

Gunma University

Hideki Kambara

Kazunori Okano

Satoshi Takahashi

Keiichi Nagai

Chihiro Uematsu

Tomoharu Kajiyama

Takashi Anazawa

Kunio Harada

Masao Kamahori

Masataka Shirai

Kiyomi Taniguchi

Tetsuo Nishikawa

Takashi Yamada

Hisanori Nasu

Maiko Tanabe

Hiroko Matsunaga

Sukeyoshi Tsunekawa

Takashi Irie

Sumio Yamaguchi

Tamotu Simada

Guohua Zhou

Kenji Yasuda

Minoru Sakairi

Yoshitoshi Ito

Masato Mitsuhashi

Yoshiaki Yazawa

Tomoaki Sumitani

Yoshinori Harada

Hiroaki Machida

Jirou Tokita

Hiroyuki Tsunoda

Hiroko Furuyama

Kazuko Kawamoto

Ken Takei

Mitsuo Usami

Hideki Hasegawa

Kazumichi Imai

Kenichi Watanabe

Ken Ninomiya

Yuji Miyahara

Yoshinobu Kohara

Masayasu Kuwahara

Jiro Tokita

Akihiko Kishimoto

Ken'ichi Watanabe

Toru Habu

Masafumi Kawamura

Kazunon Okano

Zheng Ping Li

Yoshiko Katayama

Teruo Togusari

Kazuyuki Irisawa

Katsuhiko Murakami

Huan Huang

Setsuko Seki

Mari Ohta

Simada Tamotu

The present invention relates to a method, a device, and an apparatus for analyzing the expression of a gene in single cells. Specifically, the present invention relates to: a device for gene expression analysis, characterized by including a support, in which a nucleic acid probe having a test nucleic acid capture sequence and a known sequence, and further containing a cell recognition tag sequence which differs depending on the difference in position on the surface of the support or in the vicinity of the surface thereof, and a common primer sequence having a known sequence is two-dimensionally distributed and immobilized on the surface of the support or in the vicinity of the surface thereof; and a method and an apparatus using the device for gene expression analysis.

Tag-sequence-attached two-dimensional cDNA library device, and gene expression analysis method and gene expression analysis apparatus each utilizing same

The present invention provides a method and/or means for collecting and analyzing an individual cell in a tissue, and at the same time, quantitatively monitoring the expression levels of various genes while keeping two-dimensional information in the tissue. Specifically, the present invention provides a method comprising preparing a cDNA library from mRNA while keeping two-dimensional cellular distribution information and obtaining the gene expression levels at any site or all sites at a level of single cell. More specifically, the present invention provides a method comprising preparing a cDNA library in a sheet-form from mRNA while keeping two-dimensional cellular distribution information and repeatedly using the cDNA library in the detection of the gene expression, thereby allowing measurement of the expression distribution for a number of genes at a high accuracy.

Gene expression analysis ME1HOD using two dimensional cDNA library

Tag-sequence-attached two-dimensional CDNA library device, and gene expression analysis method and gene expression analysis apparatus each utilizing same

In order to conduct gene expression analysis of a number of genes in a number of cells, it has been necessary to separate cells, extract genes therefrom, amplify nucleic acids, and perform sequence analysis. However, separation of cells imposes damages on the cells, and it requires the use of an expensive system. Gene expression analysis in each cell can be carried out with high accuracy by arranging a pair of structures comprising a cell trapping section and a nucleic acid trapping section in a vertical direction to extract individual genes in relevant cells, synthesizing cDNA in the nucleic acid trapping section, amplifying nucleic acids, and analyzing the sequences using a next-generation sequencer.

Two-dimensional cell array device and apparatus for gene quantification and sequence analysis

In order to decode arbitrary sequence regions for a large number of genes in a large number of cells, it is necessary to fragment the nucleic acids and introduced a sequence, which differs for each the cell, in the respective fragments. However, in conventional constructions for analyzing large numbers of cells, there was the problem that the cleaved fragments of different regions were intermingled before a tag sequence unique to each region could be introduced. The present invention is constructed to also comprise a genetic analysis system, when trapping nucleic acids extracted from a cell in multiple regions on a substrate and synthesizing and fragmenting the complementary DNA strands (cDNA) of the nucleic acids for each individual region, for immediately introducing a tag sequence unique to each of the regions into said fragments.

Genetic analysis system

The problem to be solved by the present invention is to provide a method for preparing a sample for comprehensively and accurately analyzing gene expression in a single cell or a few cells, for example, by a large-scale DNA sequencer. The present invention relates to a method for amplifying cDNA from mRNA in a cell, comprising: (1) capturing mRNA derived from the cell by a first DNA probe containing a first tag sequence and a poly T sequence and being immobilized to a solid carrier in a single reaction vessel, and synthesizing a first strand cDNA from the mRNA by a reverse transcription reaction; (2) removing a reaction reagent from the reaction vessel while keeping the first strand cDNA synthesized onto the solid carrier in the reaction vessel; (3) adding a polynucleotide sequence consisting of one type of nucleotides to 3' terminal of the first strand cDNA on the solid carrier; (4) hybridizing a second DNA probe containing a second tag sequence and a complementary sequence to the polynucleotide sequence with the cDNA to which the polynucleotide sequence is added, and synthesizing a second strand cDNA; and (5) performing a DNA amplification reaction using the second strand cDNA synthesized on the solid carrier as a template.

Method for amplifying cDNA derived from trace amount of sample

To provide a means and a method of quickly sampling a tissue fragment from a plant tissue, quickly preserving a gene expression state within the tissue fragment, and comprehensively analyzing the gene expression state. The present invention relates to a method of sampling a plant tissue section, comprising: inserting a first gel layer into a needle; arranging a plant tissue on a second gel layer; and passing the needle through the plant tissue together with the second gel layer, and sampling a section of the plant tissue in the needle.

Plant tissue sampling method and plant gene analysis method

This invention provides a nucleic acid amplification device whereby the abundance of a target molecule can be maintained before and after a step of separately amplifying a sample such that highly accurate analysis results that can be applied to gene expression analysis can be obtained. Also, a nucleic acid amplification device having a structure in which a plurality of minute reaction cells each comprising a set of a bead-retaining space capable of retaining a single analysis bead and a reagent reaction space retaining no bead but having a volume that is large enough to cause a reagent reaction therein are positioned so as to form a planar face is provided.

Nucleic acid amplification device

Accurate and sensitive sequencing in pyrosequencing is achieved by allowing complementary strand synthesis reaction to proceed homogeneously and completely in a short time while performing luminescence reaction for a sufficiently long time. DNA as a sequencing target is immobilized on the surface of a solid support. Nucleic acid substrates are injected from a dispenser to the support site where complementary strand synthesis is in turn performed rapidly and completely in a short time under a small reaction volume. Next, the support together with the product thereon is moved into a luminescence reaction solution where luminescence reaction is in turn performed. Thus, a DNA complementary strand synthesis reaction site and a luminescence reaction site are completely separated. The support surface is also washed by dipping the support in the luminescence reaction solution that contains a luminescence reagent and an enzyme that degrades redundant nucleic acid substrates.

DNA analysis apparatus

Provided is a nucleic acid substrate which has nucleic acid substrate characteristics equivalent to those of dATP, has a low substrate specificity for luciferase, exerts no negative effect on enzymatic reactions such as a complementary-strand synthesis, and therefore is particularly suitable for the pyrosequencing method. As a nucleic acid substrate complementary to nucleotide T, a 7-substituted deoxyribonucleotide triphosphate whose 7-position of a purine group is modified by a substituent is used as a substitute for a nucleotide &#x3b1;-thiotriphosphate analog.

Method and reagent for gene sequence analysis

It is an object to provide a method of suitably analyzing the amount of gene expression of a single-cell. A method of detecting a nucleic acid comprising

Methods for quantitative cDNA analysis in single-cell

It is intended to provide a novel convenient approach for DNA quantitative analysis that overcomes the disadvantages of conventional formulations. A standard DNA sample is prepared by introducing a single-base substitution into target DNA, and a predetermined amount thereof is mixed with a target DNA sample. The target and standard DNAs are amplified using the same primers designed to amplify a region comprising the single-base substitution site. To a hybridization product of a probe capable of binding to a site immediately before the single-base substitution site, ddATP, ddGTP, ddCTP, and ddTTP are sequentially added one by one to perform a complementary strand synthesis reaction. Luciferase reaction-induced luminescence derived from the formed pyrophosphoric acid is detected. The target DNA is quantitated from the amount of the detected luminescence and the amount of the added standard DNA sample.

Method for nucleic acid quantitation

By the conventional technique for dispensing more than one reagents accurately, the system is complicated and thus a compact and inexpensive system is difficult to realize. In the present invention, the pressurized dispensing system utilizing a capillary is realized, and in addition, in order to reduce the leakage of reagents different from the reagent dispensed, by forming air layers at the tips of the capillaries after dispensing, a compact, simple, inexpensive analysis apparatus is realized.

Small size gene analysis apparatus

This invention relates to: the development of a mutant firefly luciferase in order to use dATP as a DNA polymerase substrate upon pyrosequencing, such luciferase being subjected to substrate specificity modification in a manner such that the dATP-induced activity alone is decreased while the ATP-induced activity is maintained; and a mutant firefly luciferase for which the proportion of activity induced by dATP to activity induced by ATP (dATP/ATP) is lower than that for the wild-type firefly luciferase, in which an amino acid identified based on homology analysis as corresponding with the 421amino acid (glycine) of the amino acid sequence of the wild-type North American firefly () luciferase has been substituted with a polar amino acid.

Mutant firefly luciferase

Growing community of inventors

Hachioji, Japan

Hideki Kambara