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Method for constructing DNA sequencing library of genome under detection and application of method

A DNA sequencing and sequencing library technology, applied in the biological field, can solve the problems of limited antibody quality and inability to obtain effective histone modification information, so as to avoid local damage and improve recognition efficiency

Active Publication Date: 2016-07-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chromatin immunoprecipitation technology is extremely limited by many factors such as the number of cells and the quality of antibodies. If a sufficient amount of DNA for library construction cannot be obtained, effective histone modification information cannot be obtained.
The traditional chromatin immunoprecipitation technique includes several important steps such as crosslinking, breaking DNA, antibody incubation, uncrosslinking and eluting DNA. However, the traditional chromatin immunoprecipitation technique is usually used to study easily obtained, order of magnitude in 10 6 The above common types of cells are powerless for the study of this extremely difficult to obtain and a small number of types of cells in the early stages of embryonic development

Method used

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  • Method for constructing DNA sequencing library of genome under detection and application of method
  • Method for constructing DNA sequencing library of genome under detection and application of method
  • Method for constructing DNA sequencing library of genome under detection and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Construction of DNA sequencing library of cell genome

[0089] 1.1 Reagent preparation

[0090] Lysis buffer

[0091] 0.5% NP-40

[0092] 0.5%Tween

[0093] 0.1% SDS

[0094] MNase working buffer (MNase working buffer)

[0095] ·100mM Tris-HClph8.0

[0096] 2mM CaCl2

[0097] MNase Dilute buffer (MNaseDilutebuffer)

[0098] ·50mM Tris‐HCl, pH8.0

[0099] 1mM CaCl2

[0100] 0.2% Triton X-100

[0101] 10xMNase stop buffer (10xMNaseStopbuffer)

[0102] 110mM Tris-HCl pH8.0

[0103] 55mM EDTA

[0104] 2xRIPA buffer2xRIPAbuffer(onice)

[0105] 280mM NaCl

[0106] 1% Triton X‐100

[0107] 0.1% SDS

[0108] 0.2% Na‐Deoxycholate

[0109] 5mMEGTA

[0110] RIPA buffer RIPAbuffer

[0111] ·10mM Tris pH8.0

[0112] 1 mM EDTA

[0113] 140mM NaCl

[0114] 1% Triton X‐100

[0115] 0.1% SDS

[0116] 0.1% Na‐Deoxycholate

[0117] Lithium chloride buffer (LiClwashbuffer)

[0118] 250mMLiCl

[0119] ·10mM Tris pH8.0

[0120] 1 mM EDTA

[0121] 0.5% N...

Embodiment 2

[0137] Example 2 Screening of dephosphorylation enzymes

[0138] In order to obtain the best dephosphorylated end repair results and ensure the final DNA yield, the inventors screened the most suitable dephosphorylation treatment enzyme from the three combinations of T4PNK, T4DNA polymerase, Klenow or T4PNK or rSAP. The specific experimental process is as follows Said:

[0139] The mouse embryonic stem cells grown on the six-well cell culture plate were taken out of the incubator, the medium was sucked off, and the medium was gently washed with 1ml of PBS to wash off the residual medium. Then add 0.5ml of trypsin with a concentration of 0.05%, digest at 37° for three minutes, and stop the digestion reaction with 1ml of fresh medium. In order to make the cells completely become a single suspended cell state, pipette repeatedly with a 1ml large pipette until no cell clumps are visible to the naked eye. Then centrifuge at a low speed of 2000rmp for 5 minutes. After the superna...

Embodiment 3

[0159] In order to verify the effectiveness of the library construction method proposed in this application, the inventors adopted the most stringent testing method: through co-immunoprecipitation DNA sequencing technology based on a large number (1x10e7) of mouse embryonic stem cells (1x10e7) in the ENCODE database (ChIP-sequence) results were compared to see the correlation between the two.

[0160] Specifically, the inventor compared the map data obtained by the inventor with the ENCODE map data, and loaded it into UCSCbrowser. Then, by observing the peak distribution of H3K4me3 and combining its positional relationship with the gene promoter, the inventor judged the Whether the method for building the database proposed by the invention is successful.

[0161] The result is as Figure 16 As shown, it can be seen from the results (genomic position: chr6:51,271,690-52,394,589), whether it is the result (ENCODE) obtained from millions of cells or the ChIP-sequence result of t...

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Abstract

The invention provides a method for constructing a DNA sequencing library of a genome under detection, comprising the steps: (1), subjecting the genome under detection to first digestion by using micrococcal nuclease to obtain a first digestion product; (2), subjecting the first digestion product to co-immunoprecipitation to obtain a co-immunoprecipitation product; (3), subjecting the co-immunoprecipitation product to second digestion by using protease K to obtain second digestion product; (4), subjecting the second digestion product directly to dephosphorylation by using shrimp alkaline phosphatase to obtain a dephosphorylation product; (5), subjecting the dephosphorylation product directly to denaturalization to obtain a denaturalization product with single-branched DNA molecules; and (6), acquiring the sequencing library by TELP (tailing, extension, ligation and PCR) method based on the denaturalization product with single-branched DNA molecules.

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 a genome to be tested, a device for constructing a DNA sequencing library of a genome to be tested, a method for determining DNA sequence information of a genome to be tested, and a system for determining DNA sequence information of a genome to be tested And a method for determining the sequence information of the chromatin target region of the genome to be tested. Background technique [0002] Recent studies in the field of epigenetics have shown that histone modification plays a vital role in maintaining cell growth and development. Therefore, how to obtain systematic and comprehensive histone modification information of different types of cel...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C40B60/14C12Q1/68
CPCC12N15/10C12Q1/68C40B50/06C40B60/14C12N15/1093C12Q1/6806
Inventor 颉伟张冰洁
Owner TSINGHUA UNIV
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