Method and application of constructing dna sequencing library of genome to be tested

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: 2019-03-29
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 and application of constructing dna sequencing library of genome to be tested
  • Method and application of constructing dna sequencing library of genome to be tested
  • Method and application of constructing dna sequencing library of genome to be tested

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

[0095] 100mM Tris-HCl pH8.0

[0096] 2mM CaCl2

[0097] MNase Dilute buffer

[0098] 50mM Tris‐HCl, pH 8.0

[0099] 1mM CaCl2

[0100] 0.2% Triton X‐100

[0101] 10x MNase Stop buffer (10xMNase Stop buffer)

[0102] 110mM Tris-HCl pH 8.0

[0103] 55mM EDTA

[0104] 2x RIPA buffer2xRIPA buffer(on ice)

[0105] 280mM NaCl

[0106] 1% Triton X‐100

[0107] 0.1% SDS

[0108] 0.2% Na‐Deoxycholate

[0109] 5mM EGTA

[0110] RIPA buffer RIPA buffer

[0111] 10mM Tris pH 8.0

[0112] 1 mM EDTA

[0113] 140mM NaCl

[0114] 1% Triton X‐100

[0115] 0.1% SDS

[0116] 0.1% Na‐Deoxycholate

[0117] LiCl wash buffer

[0118] 250mM LiCl

[0119] 10mM Tris pH 8.0

[0120] 1 mM EDTA

[0121] 0.5% NP‐40 (now known as Igepal CA‐630)

[0122] 0.5% Na-deoxy...

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, on the one hand, the inventor compared the map data obtained by the inventor with the ENCODE map data, and loaded it into the UCSC browser, and then judged by observing the peak distribution of H3K4me3 and combining its positional relationship with the gene promoter. Whether the method for building a database proposed by the present 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-sequen...

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Abstract

Provided is a method for constructing a DNA sequencing library for a to-be-detected genome. The method comprises: (1), carrying out first digestion processing on a to-be-detected genome by using micrococcal nuclease, so as to obtain a first digestion processing product; (2), carrying out co-immunoprecipitation processing on the first digestion processing product, so as to obtain a co-immunoprecipitation processing product; (3), carrying out second digestion processing on the co-immunoprecipitation processing product by using protease K, so as to obtain second digestion processing product; (4), directly carrying out dephosphorylation processing on the second digestion processing product by using shrimp alkaline phosphatase, so as to obtain a dephosphorylation processing product; (5), directly carrying out denaturalization processing on the dephosphorylation processing product, so as to obtain a denaturalization processing product containing single-stranded DNA molecules; and (6), based on the denaturalization processing product containing single-stranded DNA molecules, obtaining a sequencing library according to a TELP method.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C40B50/06C40B60/14C12Q1/6806
CPCC12N15/10C12Q1/68C40B50/06C40B60/14
Inventor 颉伟张冰洁
Owner TSINGHUA UNIV
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