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Method for identification and monitoring of epigenetic modifications

A biological sample and imprinting technology, applied in the field of identification and monitoring of epigenetic modifications, can solve the problems of little progress and limitations

Inactive Publication Date: 2009-10-07
NIMBLEGEN SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this approach is limited by the proximity of genes to known imprinted genes, which are relatively small in number because only a small fraction of human genes are genomically imprinted
[0010] Despite the public health importance of imprinted genes and the considerable effort expended by many laboratories, relatively little progress has been made in developing methods for the identification of new imprinted genes

Method used

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  • Method for identification and monitoring of epigenetic modifications
  • Method for identification and monitoring of epigenetic modifications
  • Method for identification and monitoring of epigenetic modifications

Examples

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Embodiment 1

[0067] Example 1: ChIP Chip for Discovery and Monitoring of Imprinted Genes TM

[0068] To determine genomic regions containing imprinted genes, applicants performed a ChIP Chip TM Experimentally, regions with overlapping open and closed chromatin marks were identified by their association with preferred modified histone marks, such as AcK9H3, 3MeK9H3, 2MeK9H3 and RNA polymerase II.

[0069] In short, to perform ChIP Chip TM In the experiment, cells were chemically treated to cross-link DNA-binding proteins at their binding sites. DNA was isolated from these cells, fragmented, and enriched by immunoprecipitation with antibodies against modified histones and the RNA polymerase of interest (see generally Ng et al., Genes Dev. 16, 806-819 (2002); Wyrick et al., Science 290, 2306-2309 (2002); Weinmann et al., Genes Dev. 16, 235-244 (2002)).

[0070] These enriched DNAs were then amplified by LM-PCR. Such as figure 1 As shown, the methods of the present invention are not limi...

Embodiment 2

[0073] Example 2: Microarray Analysis of Coexisting Silent and Active Chromatin Marks as a High-Throughput Method to Identify Newly Imprinted Genes

[0074] Regardless of the CpG island methylation status in a particular cell type, Applicants performed a series of microarray experiments in the presence or absence of CpG island or GC-rich sequences to efficiently screen or identify concurrent "active" or " Silent" chromatin marks.

[0075] Chromatin immunoprecipitation

[0076] Chromatin immunoprecipitation (ChIP) was performed in cultured HeLA and 293 cells using the known ChIP protocol electronically published by Farnham Laboratories, published on December 25, 2005 as follows: http: / / genomecenter.ucdavis.edu / farnham / farnham / protocols / chips.html , a protocol incorporating ligation-mediated PCR (LMPCR) based on the method of Ren et al. ((2000) Science 290, p. 2306) (both articles are incorporated herein by reference in their entirety).

[0077] Materials for ChIP analysis ...

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Abstract

The present invention provides novel methods for identifying and monitoring epigenetic modifications, such as imprinted genes, using microarray based technology. Specifically, the invention detects imprinted genes by the presence of overlapping closed and open chromatin markers. The invention also discloses a method for detecting the loss of imprinting on a genome-wide scale, which is indicative of a variety of medical conditions. Diagnostic assays and chromatin structure markers for identifying gene imprinting and loss thereof are also disclosed.

Description

technical field [0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application 60 / 749924, filed December 13, 2005, which is incorporated herein by reference in its entirety. [0003] Statement on federally funded research and development Not applicable. Background technique [0004] According to the traditional laws of Mendelian genetics, humans inherit two copies of their genes - one from the mother and one from the father. Normally, both copies of each gene are active or "turned on" inside the cell. However, in some cases, only one of the two copies opens normally. Which copy is active depends on the parental source. Some genes are only active when they are inherited from the father, while others are only active when they are inherited from the mother. This phenomenon is called "genomic imprinting". The parental source of genes undergoing genomic imprinting tends to be genetically marked (ie, through the proces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/112C12Q2600/156C12Q2600/154C12Q1/6809C12Q1/6883C12Q1/6837C12Q2522/101C12Q2523/101C12Q2537/164C12Q2565/501
Inventor R·D·格林A·P·范伯格H·T·比约恩森
Owner NIMBLEGEN SYST
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