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METHOD FOR ISOLATING SPECIFIC GENOMIC REGION USING MOLECULE BINDING SPECIFICALLY TO ENDOGENOUS DNA SEQUENCE 新技術説明会

外国特許コード F150008421
掲載日 2015年10月9日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2013JP074107
国際公開番号 WO 2014125668
国際出願日 平成25年9月6日(2013.9.6)
国際公開日 平成26年8月21日(2014.8.21)
優先権データ
  • 特願2013-026310 (2013.2.14) JP
  • 特願2013-143282 (2013.7.9) JP
発明の名称 (英語) METHOD FOR ISOLATING SPECIFIC GENOMIC REGION USING MOLECULE BINDING SPECIFICALLY TO ENDOGENOUS DNA SEQUENCE 新技術説明会
発明の概要(英語) A method that comprises: (A) a step for fragmenting a genomic DNA that maintains an interaction state with a molecule under the interaction; and (B) a step for contacting the genomic DNA with a foreign molecule binding to a specific endogenous DNA sequence in the genomic DNA. According to this method, an arbitrary genomic region can be specifically isolated while maintaining an interaction state with a molecule under the interaction by using an endogenous DNA sequence within a target genomic region or the neighborhood thereof in a cell to be analyzed, without requiring a procedure for inserting a recognition sequence of a foreign DNA-binding molecule into the neighborhood of the target genomic region of the cell to be analyzed.
従来技術、競合技術の概要(英語) BACKGROUND ART
Biochemical and molecular biological analysis of chromatin region, the functional expression of the genome in order to understand the molecular mechanisms of very important. However, still the chromatin region of the biochemical properties are not sufficiently analyzed. This is because, the chromatin region of biochemical and molecular biological methods can be used for analyzing the sample is limited it is believed that the cause.
Biochemical and molecular biology of the chromatin region in order to perform the analysis, genomic DNA and genomic DNA molecules interact with the interaction state holding the specimen needs to be subjected to analysis., The ejector @num@ in the same manner as the frequency of occurrence of turbulence in the diffuser @num@ can be reduced more reliably, in a more reliable energy conversion efficiency of the ejector @num@ can be increased, pressure of the hydraulic oil output from the diffuser @num@ (energy) can be increased.
(i) chromatin immunoprecipitation method chromatin immunoprecipitation method (hereinafter, 'ChIP method' is referred.) Is, using antibodies against known DNA binding protein immunoprecipitated with specific genomic regions, in the method of isolation of the region (non-patent document 1, reference 2). Therefore, ChIP method, if there is no information about the quality DNA binding protein can be used is a limitation. Also, in general, a plurality of genomic DNA DNA binding protein for binding to the region, in a variety of immunoprecipitates are mixed and the genomic region, genomic region ChIP method specified by the problem that it is difficult to isolate only has. Further, ChIP method, known DNA binding protein can be isolated genomic regions are not bound to a problem that the.
(Ii) Chromosome Conformation Capture method (hereinafter referred to as' method 3C ' is referred.) Or a derivative thereof in the method 3C, interacting with specific genomic region can be identified genomic regions (see non-patent document 3-5). However, method 3C, the enzyme reaction and restriction enzyme DNA ligase, such as under a non-cross-linking because it has to be not in optimal conditions, the interaction is detected non-physiological high probability that the subject is a problem. In addition, cross-linked under for imperfections in the restriction enzyme treatment, in a region adjacent to the target genome is very high and the background signal is amplified by PCR, detection of an interaction of the unknown is difficult.
(Iii) Fluorescence in situ Hybridization method (hereinafter, 'FISH method' is referred.) FISH method alone, or can be combined with a fluorescence antibody method, or another specific RNA genome region of the genomic region, the interaction of the protein can be detected. However, this method is a low resolution, in addition, the interaction of the unknown molecules of cannot be detected.
Method (iv) Proteomics of Isolated Chromatin segments (hereinafter 'PICh method' is referred.) PICh method, using nucleic acid probes specific for, and methods for isolating specific genomic regions, a large number of the probe is complementary to telomeric regions consisting of a repeated sequence shown can be isolated (see non-patent document 6). However, cross-linked PICh method under the probe and the target nucleic acid of the genomic region and annealing is needed, specific low copy number or copy 1 determines whether or not the isolation of the genomic region is not shown.
(v) Non-patent document 7 is of the non-patent document 7, isolated genomic regions identified by affinity purification as described attempts. However, the cell is used, yeast, higher eukaryotic cells includerelease of has not been performed. In addition, by utilizing a system Cre-loxP, specific genomic regions are cut out, with this operation, a change in chromatin structure might be. In addition, cannot be processed for cross-linked with formaldehyde, in the course of purification of the protein or DNA is bonded to the molecule such as the possibility of dissociation.
(vi) IChIP method developed by the inventors of the present invention is a method (patent document 1, non-patent document 8, reference 9). While holding the physiological chromatin structure to isolate specific genomic regions, to perform the following operation. (1) Analysis of target cells to the exogenous DNA region near the target genomic region recognition sequences are inserted into the binding molecules. (2) DNA binding domain and an exogenous DNA binding molecules, such as with the existing antibodies or other protein which is recognized by the tag-fusion molecule, be expressed in a cell to be analyzed. (3) The cells were cross-linked with formaldehyde or the like as necessary. By this operation, the target genome that interact with that region protein, RNA, DNA and other molecules are cross-linked. In addition, by this operation, a binding molecule tagged exogenous DNA recognition sequence is inserted into the cross linked. (4) Then, the cell is, cross-linked DNA with a restriction enzyme cleaving enzyme DNA such as by fragmenting or ultrasonication. (5) Foreign DNA tagged complex including a binding molecule, an antibody recognizing the tag or protein or the like is fixed to the sedimented using a carrier. (6) Precipitated complex molecules interact with the target genomic region is analyzed. As a problem of iChIP method, the vicinity of the target genome analysis of target cells exogenous DNA recognition sequence of the binding molecule for an operation to insert takes more time, the insertion of the recognition sequence and having a negative impact on the interaction and cannot be removed.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • OSAKA UNIVERSITY
  • 発明者(英語)
  • FUJII, Hodaka
  • FUJITA, Toshitsugu
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
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