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Bacterial HI-C genome and plasmid conformation capturing method

A HI-C and genomic technology, applied in biochemical equipment and methods, combinatorial chemistry, recombinant DNA technology, etc., can solve problems such as error-prone and immature, and achieve highly practical results

Pending Publication Date: 2021-08-06
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the current Hi-C chromosome conformation capture technology is immature and error-prone, especially the conformation capture method of bacterial genomes and plasmids

Method used

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  • Bacterial HI-C genome and plasmid conformation capturing method
  • Bacterial HI-C genome and plasmid conformation capturing method
  • Bacterial HI-C genome and plasmid conformation capturing method

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Experimental program
Comparison scheme
Effect test

Embodiment

[0040] The first step: bacterial culture.

[0041] 1. Dip a small amount of bacterial solution with an inoculation loop, streak it on an LB agar plate (or an identification agar plate), and place the plate in a 37°C incubator for 18-24 hours.

[0042] 2. Cultivate the single bacterium in 50 mL of LB broth at 37°C on a shaker for 3 hours and 15 minutes.

[0043] 3. Collect all the bacterial liquid, centrifuge at 5000 rpm at 4°C for 8 minutes, discard the culture liquid, and collect the bacterial precipitate.

[0044] 4. Add 6mL of fresh LB broth, resuspend the bacteria, and wash. Then, centrifuge at 5000 rpm for 8 minutes at 4°C to collect the bacterial pellet.

[0045] The second step: cross-linking reaction.

[0046] 1. Prepare 20mL of 3% formaldehyde solution (protected from light on ice): 3% formaldehyde solution

[0047] 18.4mL 1*PBS solution + 1.6 / mL 37% w / v formaldehyde solution

[0048] 2. Add the prepared 3% formaldehyde solution into a 50mL centrifuge tube contai...

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Abstract

The invention discloses a bacterial HI-C genome and plasmid conformation capturing method. The method comprises the following capturing steps: bacterial culture, cross-linking reaction, sample integrity verification, bacterial lysis, chromatin digestion, biotin filling, ligation reaction, reverse cross-linking, library extraction, biotin removal, phenol chloroform DNA extraction and second generation library establishment. According to the method, DNA and protein are crosslinked together through formaldehyde treatment so as to fix the conformation of the DNA, enzyme digestion, biotin introduction, enzyme linkage and extraction are carried out, then library construction and high-throughput sequencing are carried out on treated nucleic acid, and finally, interaction information between bacterial chromatin fragments and between bacterial chromatin fragments and plasmids can be revealed by analyzing sequencing data. The method can be applied to genome assembly, gene expression regulation research and the like.

Description

technical field [0001] The invention relates to the technical field of chromosome conformation capture, in particular to a bacterial HI-C genome and plasmid conformation capture method. Background technique [0002] The main step of Hi-C is to prepare a 3C template suitable for Hi-C, cut with restriction enzymes, and connect biotin-labeled nucleotides to the end. DNA purification followed by blunt-end ligation and shearing. Biotin PuLL-down ensures that only ligated fractions are used for analysis. Sequenced short-read sequence Reads are mapped to the genome, and when the pair of short-read sequences are found on different fragments, the interacting fragments are scored accordingly. In this way, a junction frequency matrix containing all segments of the genome is constructed. The 4C determination test can be verified in Hi-C. Hi-C experiments let scientists discover that there are specific chromatin compartments in chromatin, and chromatin and heterochromatin are in diff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12N15/1003C12N15/1013C12Q1/6806C12Q1/6869C40B50/06C12Q2535/122
Inventor 连新磊冯晓茵李梦圆孙坚刘雅红
Owner SOUTH CHINA AGRI UNIV
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