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Method for constructing DNA (deoxyribonucleic acid) sequencing library of to-be-detected genome and application thereof

A DNA sequencing and genome technology, applied in the biological field, can solve the problems of limited cell number, inability to obtain effective and high-resolution genome three-dimensional structure information, etc.

Inactive Publication Date: 2017-09-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genome-wide chromatin conformation capture technology is extremely limited by the number of cells. If a sufficient amount of DNA containing enough effective ligation events for library construction cannot be obtained, it is impossible to obtain effective and high-resolution genome three-dimensional structure information of

Method used

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  • Method for constructing DNA (deoxyribonucleic acid) sequencing library of to-be-detected genome and application thereof
  • Method for constructing DNA (deoxyribonucleic acid) sequencing library of to-be-detected genome and application thereof
  • Method for constructing DNA (deoxyribonucleic acid) sequencing library of to-be-detected genome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of DNA sequencing library of cell genome

[0051] 1.1 Reagent preparation

[0052] Lysis buffer

[0053] 10mM Tris-HCl., pH=7.4

[0054] 10mM NaCl

[0055] 0.5% NP-40

[0056] 0.1mM EDTA

[0057] 1X Proteinase Inhibitor

[0058] 2X biotin and streptavidin magnetic bead binding solution (2X Binding buffer)

[0059] 10mM Tris-HCl., pH=8.0

[0060] 2M NaCl

[0061] 1 mM EDTA

[0062] Tween wash buffer

[0063]5mM Tris-HCl., pH=8.0

[0064] 1M NaCl

[0065] 0.05%Tween

[0066] 0.5mM EDTA

[0067] 1.2 Formaldehyde crosslinking

[0068] Transfer the collected samples to a freshly prepared PBS solution containing 1% formaldehyde under a stereoscope with a mouth pipette, fix at room temperature for 10 minutes, add 2.5M glycine solution to a final concentration of 0.2M, and let stand at room temperature for 10 minutes. Use a mouth pipette to transfer the sample into the PBS solution to wash once, then transfer to a PCR tube.

[0069] 1.3 Cl...

Embodiment 2

[0086] In order to compare the sisHi-C of the present application with the previous method (Rao et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatinlooping. Cell. 2014) in the effect of a small number of cells, the inventor used a mouth pipette to accurately Four groups of mouse embryonic stem cells were counted. The number of cells in each group was 500. Then, the inventors used sisHi-C and previous methods to build banks for two groups of cells (respectively labeled as repeated experiment 1 and repeated experiment 2). The concentration of DNA was measured before PCR, and the proportion of effective data was analyzed after library sequencing. Figure 8 The results of A show that before PCR, the DNA concentration of the two groups using the sisHi-C method is significantly higher than that of the two groups using the previous method; Figure 8 The results of B show that sisHi-C significantly reduces the loss of DNA. In the sequencing data...

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Abstract

The invention provides a method for constructing a DNA (deoxyribonucleic acid) sequencing library of a to-be-detected genome. The method comprises the following steps of (1) utilizing restriction enzyme to digest the to-be-detected genome, so as to obtain a digested product; (2) marking the digested product by biotin, so as to obtain a biotin-marked product; (3) linking the biotin-marked product by DNA ligase, so as to obtain a linked product; (4) decrosslinking the linked product; (5) purifying the decrosslinked product; (6) performing ultrasonic and precipitation treatment on the purified product, wherein the precipitation treatment is performed via contact between the ultrasonic product and streptavidin magnesphere so as to obtain a target DNA segment bonded with the streptavidin magnesphere; and (7) constructing the library on the basis of the target DNA segment bonded with the streptavidin magnesphere.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for constructing a DNA sequencing library of a genome to be tested and its application. More specifically, the present invention relates to a method for constructing a DNA sequencing library of the genome to be tested, a method for determining the DNA sequence information of the genome to be tested, and a method for determining the three-dimensional spatial structure of the genome to be tested. Background technique [0002] With the development of genomics in recent years, the Human Genome Project successfully mapped the human genome DNA sequence. According to the research related to the Human Genome Encyclopedia Project, tens of thousands of genes and hundreds of thousands of different gene regulatory elements were found through analysis. Recent studies have shown that the three-dimensional structure of the genome has an important imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/68
Inventor 颉伟杜振海
Owner TSINGHUA UNIV
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