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Chromosome conformation capture method including selection and enrichment steps

A subset and selection technique, applied in the field of subsetting interacting nucleic acid segments, identifying one or more interacting nucleic acid segments indicative of a specific disease state, can solve complex and other problems

Active Publication Date: 2016-06-08
BABRAHAM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While this provides information about all interactions occurring within the nuclear composition at a particular point in time, there are approximately 800,000 ligatable fragments per cell (e.g., if there is a restriction of 6 base pairs of recognition sites Endonucleases such as HindIII or BglII have been used for Hi-C library generation), which makes the library too complex and prevents the interrogation of specific interactions between individual regions

Method used

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  • Chromosome conformation capture method including selection and enrichment steps
  • Chromosome conformation capture method including selection and enrichment steps
  • Chromosome conformation capture method including selection and enrichment steps

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1: Promoter Capture Hi-C Protocol

[0120] Day 1: Fixation of cells, HindIII digestion

[0121] 1. Use 3x10 7 -4x10 7 cells start. Make 37ml in room temperature DMEM / 10%FBS.

[0122] 2. Add formaldehyde to a final concentration of 2% and fix at room temperature for exactly 10 minutes while mixing on a rocking device.

[0123] 3. Quench the reaction by adding 6 ml of cold 1M glycine (final 0.125M).

[0124] 4. Incubate for 5 minutes at room temperature, followed by 15 minutes on ice.

[0125] 5. Centrifuge at 1500 rpm (400xg) for 10 minutes at 4°C.

[0126] 6. Discard the supernatant, carefully resuspend the pellet in cold 1xPBS, and add cold 1xPBS to a final volume of 50ml.

[0127] 7. Centrifuge at 1500 rpm (400xg) for 10 minutes at 4°C, then discard the supernatant. Cells can be snap frozen in liquid nitrogen and stored at -80°C.

[0128] 8. Resuspend cells in 50 ml of ice-cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% IgepalCA-630, and add ...

Embodiment 2

[0352] Example 2: Sequence capture of regions interacting with bait loci (SCRiBL) Hi-C protocol

[0353] This study describes the following three variants of SCRiBLHi-C, two of which (b and c below) allow sample barcoding and multiplexing:

[0354] a) Regular SCRiBLHi-C (unbarcoded)

[0355] b) SCRiBLHi-C barcoded before capture

[0356] c) SCRiBLHi-C barcoded after capture.

[0357] From the 1st to the 10th day according to the scheme in embodiment 1, then continue to carry out the following steps:

[0358] a) Conventional SCRiBLHi-C:

[0359] Follow the protocol in Example 1 from the 11th to the 13th day, and then proceed to step 14 below.

[0360] b) Capture pre-barcoded SCRiBLHi-C:

[0361] Day 11: Biotin-streptavidin pulldown and adapter ligation

[0362] 1. Quant-iTPicoGreen (Life Technologies P7589) assay was used to determine the yield of DNA. Prepare 1:20 and 1:50 dilutions of the samples. The expected yield from 40 μg of starting material is approximately 1...

Embodiment 3

[0705] Example 3: Research on MYC Gene Promoter Interaction

[0706] The methods described herein allow simultaneous capture of chromosomal interactions for thousands of promoters, which can then be individually analyzed. As an example, genomic interactions around the MYC gene are shown here. The results can be referred to as shown in this paper figure 2 to explain.

[0707] figure 2 (A) shows the genomic region surrounding the MYC gene. figure 2 (A) Shows the location of regions interacting with the MYC promoter in CD34+ hematopoietic progenitor cells, as identified by the methods of the present invention. Asterisks indicate the position of the interacting region located 1.8 Mb downstream of the gene identified as described below.

[0708] figure 2 (B) Example of DNAFISH showing CD34+ nuclei. In these experiments, for regions in the MYC locus (such as figure 2 (A) The location indicated by the middle bar) specific fluorescently labeled DNA probes hybridized to th...

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Abstract

The invention relates to a method for identifying nucleic acid segments which interact with a target nucleic acid segment by use of an isolating nucleic acid molecule, and to kits for use in said method. The invention also relates to a method of identifying one or more interacting nucleic acid segments that are indicative of a particular disease state.

Description

field of invention [0001] The present invention relates to methods for identifying nucleic acid segments that interact with a target nucleic acid segment, in particular for identifying nucleic acid segments that interact with a subset of target nucleic acid segments, and reagents for performing the method box. The invention also relates to methods of identifying one or more interacting nucleic acid segments indicative of a particular disease state. Background of the invention [0002] Regulatory elements play a key role in the genetic control of organisms and have been shown to contribute to health and disease (such as cancer and autoimmune diseases). The identification of these regulatory elements may also have potential applications in molecular biology, for example in expression systems and genetic modification techniques. However, while thousands of regulatory elements have been mapped in mouse and human genomes, determining which target genes they regulate represents ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2537/159C12Q2521/501C12Q2523/101C12Q2523/301C12Q1/6855C12Q1/6869C12Q1/6876C12Q1/6883C12Q2600/16
Inventor P.弗拉泽C.奥斯本S.舍恩费尔德
Owner BABRAHAM INST
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