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METHOD FOR ISOLATING SPECIFIC GENOMIC REGION USING MOLECULE BINDING SPECIFICALLY TO ENDOGENOUS DNA SEQUENCE commons meetings

Foreign code F150008421
Posted date Oct 9, 2015
Country WIPO
International application number 2013JP074107
International publication number WO 2014125668
Date of international filing Sep 6, 2013
Date of international publication Aug 21, 2014
Priority data
  • P2013-026310 (Feb 14, 2013) JP
  • P2013-143282 (Jul 9, 2013) JP
Title METHOD FOR ISOLATING SPECIFIC GENOMIC REGION USING MOLECULE BINDING SPECIFICALLY TO ENDOGENOUS DNA SEQUENCE commons meetings
Abstract 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.
Outline of related art and contending technology 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.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 相互作用している分子との相互作用状態を保持したまま特定ゲノム領域を単離する方法であって、以下の工程(A)および(B)を含むことを特徴とする特定ゲノム領域の単離方法。
(A)相互作用している分子との相互作用状態を保持しているゲノムDNAを断片化する工程
(B)ゲノムDNA中の特定の内在性DNA配列に結合する外来性分子と、ゲノムDNAとを接触させる工程

[請求項2]
 さらに、(C)ゲノムDNAと、当該ゲノムDNAと相互作用している分子との相互作用状態を保持する処理を行う工程を含むことを特徴とする請求項1に記載の特定ゲノム領域の単離方法。

[請求項3]
 前記外来性分子が、外来性DNA結合蛋白質、外来性核酸または外来性蛋白質-核酸複合体であることを特徴とする請求項1または2に記載の特定ゲノム領域の単離方法。

[請求項4]
 前記外来性分子が、ジンクフィンガー蛋白質、TALエフェクター蛋白質または不活性型Cas9蛋白質とガイドRNA(gRNA)の複合体であることを特徴とする請求項1または2に記載の特定ゲノム領域の単離方法。

[請求項5]
 以下の工程1~3を含むことを特徴とする請求項1~4のいずれかに記載の特定ゲノム領域の単離方法。
工程1:前記外来性分子と、細胞内のゲノムDNAとを接触させる工程
工程2:相互作用している分子との相互作用状態を保持している前記細胞内のゲノムDNAを断片化する工程
工程3:前記外来性分子と特異的に結合する分子を、外来性分子が結合しているゲノムDNA断片に結合させて複合体を生成し、当該複合体を回収する工程

[請求項6]
 前記外来性分子が、1種以上のタグ配列を含む融合分子であることを特徴とする請求項5に記載の特定ゲノム領域の単離方法。

[請求項7]
 前記外来性分子が、核移行シグナルを含む融合分子であることを特徴とする請求項5または6に記載の特定ゲノム領域の単離方法。

[請求項8]
 工程1において、解析対象細胞に、前記外来性分子をコードする遺伝子を導入し、当該細胞内で発現させることを特徴とする請求項5~7のいずれかに記載の特定ゲノム領域の単離方法。

[請求項9]
 以下の工程I~IIIを含むことを特徴とする請求項1~4のいずれかに記載の特定ゲノム領域の単離方法。
工程I:相互作用している分子との相互作用状態を保持しているゲノムDNAを断片化する工程
工程II:前記外来性分子と、断片化した前記ゲノムDNAとを接触させる工程
工程III:前記外来性分子に結合したゲノムDNA断片を回収する工程

[請求項10]
 工程IIにおいて、担体に固定化された前記外来性分子と、断片化した前記ゲノムDNAとを接触させることを特徴とする請求項9に記載の特定ゲノム領域の単離方法。

[請求項11]
 工程IIにおいて、前記外来性分子と断片化した前記ゲノムDNAとを接触させた後に、前記外来性分子を担体に固定化することを特徴とする請求項9に記載の特定ゲノム領域の単離方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • OSAKA UNIVERSITY
  • Inventor
  • FUJII, Hodaka
  • FUJITA, Toshitsugu
IPC(International Patent Classification)
Specified countries 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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