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Method for constructing sequencing library based on single-stranded dna molecule and its application

A DNA molecule, sequencing library technology, applied in DNA preparation, recombinant DNA technology, library and other directions, can solve problems to be improved, and achieve the effect of keeping genetic information intact, efficient determination, and less sample loss

Active Publication Date: 2018-04-20
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current next-generation sequencing technology still needs to be improved in the method of constructing gene libraries.

Method used

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  • Method for constructing sequencing library based on single-stranded dna molecule and its application
  • Method for constructing sequencing library based on single-stranded dna molecule and its application
  • Method for constructing sequencing library based on single-stranded dna molecule and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of a sequencing library

[0100] 1. The DNA used for library construction was accurately quantified by Qubit fluorometer (Invitrogen, Q32857). For double-stranded DNA, use Qubit dsDNA HS assay kit (Invitrogen, Q32854) for quantification; for single-stranded DNA, use QubitssDNA HS assay kit (Invitrogen, Q10212) for quantification. 25pg--10ng of DNA can be used as the initial amount for library construction, and then follow the TELP standard library construction steps described below.

[0101] For a small amount of double-stranded DNA (such as ChIP-Seq DNA) that is mechanically broken (such as ultrasonically broken), first perform end repair on the 5' and 3' ends of the mechanically damaged DNA. The reaction system is: 32.6 μl DNA sample , 4 μl 10x T4ligase buffer (NEB, B0202S), 1.6 μl 10 mM dNTP mix (NEB, N0447S), 0.8 μl T4PNK (NEB, M0201S), 0.8 μl T4 DNA polymerase (NEB, M0203S) and 0.16 μl Klenow fragment (NEB, M0210S) Mix well and react at 20°...

Embodiment 2

[0127] Example 2 RNA-Seq (cDNA sequencing)

[0128] Implement the present embodiment according to the steps identical with embodiment 1, only difference is:

[0129]Total cellular RNA was extracted with TRIzol reagent (Invitrogen), purified with microPoly(A) Purist Kit (Ambion, AM1919), and excess DNA was digested with DNase I, and poly-T primer (T18) and M-MLVReverse Transcriptase (Invitrogen) were used to Perform reverse transcription. After being broken to a suitable length by ultrasound, RNaseA is added to digest the RNA template strand, leaving the cDNA strand, and the single-stranded cDNA is obtained after purification. At this point, the single-stranded cDNA can be standardized using TELP technology to build a library (single-stranded DNA can skip the step of end repair and directly perform Tailing and the following steps), thereby retaining the specificity of the strand.

Embodiment 3

[0130] Example 3ChIP-Seq (chromatin immunoprecipitation sequencing)

[0131] Implement the present embodiment according to the steps identical with embodiment 1, only difference is:

[0132] Cells used for ChIP were first cross-linked with 1% formaldehyde at 37°C for 10 min, and the cells were lysed to release the chromatin. Fragments of 200-500 bp were fragmented by ultrasound. For each ChIP reaction, add 2-5 μg of the corresponding antibody and incubate overnight at 4°C. Finally, the enriched double-stranded DNA fragments were obtained, quantified by Qubit, and a certain amount of DNA was taken for TELP standardization library construction. For the application of ChIP-Seq, the library construction steps are exactly the same as those described in Example 1. Our experiment proves that this method can be used to successfully and efficiently build a standard library for DNA as low as 25pg-1ng.

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Abstract

The invention discloses a method for constructing a sequencing library based on a single-stranded DNA molecule and its application, wherein the method for constructing a sequencing library includes: (1) forming a poly(C)n tail at the 3' end of the single-stranded DNA molecule, wherein n represents The number of base C; (2) Utilize the extension primer, based on the single-stranded DNA molecule with Poly(C)n tail, obtain the double-stranded DNA molecule, wherein, the extension primer includes H(G)m at its 3' end unit, H is base A, base T or base C, and m is the number of base G; and (3) connecting a linker at the end of the double-stranded DNA molecule away from the H(G)m unit, and connecting the The resulting ligation products are amplified, and the amplified products constitute the sequencing library. The method can effectively construct a sequencing library of single-stranded DNA molecules, especially a sequencing library of micro samples.

Description

technical field [0001] The invention relates to the field of biotechnology, especially the field of gene sequencing. Specifically, the present invention relates to a method for constructing a sequencing library based on single-stranded DNA molecules and its application. More specifically, the present invention relates to a method for constructing a sequencing library based on a single-stranded DNA molecule, a device for constructing a sequencing library based on a single-stranded DNA, a method for determining the sequence information of a single-stranded DNA molecule, a method for determining the sequence information of an RNA sample, Methods for determining sequence information for regions of interest in chromatin and methods for determining genomic methylation information. Background technique [0002] The rapid development of next-generation sequencing technology has analyzed and identified many genes of normal and pathogenic traits in humans, animals and plants, and und...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C40B40/06C12N15/10C12Q1/68
CPCC12N15/1093C12N15/1096C12Q2525/173C12Q2525/191C12Q2563/179C40B50/06C12Q1/6806C12Q1/6874
Inventor 颉伟尹强宗吴婧怡徐丰彭旭
Owner TSINGHUA UNIV
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