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Novel library construction method based on illumina sequencing platform

A sequencing platform and library construction technology, applied in the direction of library, chemical library, nucleotide library, etc., can solve the problems of DNA library sample loss, cumbersome operation process, increased cost, etc., to improve labor efficiency, reduce purification process, improve The effect on success rate

Active Publication Date: 2012-07-11
WUXI QINGLAN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this also caused the inevitable loss and waste of DNA library samples in the purification process, and there are disadvantages such as cumbersome operation process and increased cost.

Method used

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  • Novel library construction method based on illumina sequencing platform
  • Novel library construction method based on illumina sequencing platform
  • Novel library construction method based on illumina sequencing platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] 1.1 Reagents

[0077] 10×T4PNK buffer(Enzymatics), dNTP(Enzymatics), T4 DNA polymerase(Enzymatics), T4PNK(Enzymatics), Klenow Fragment(3'to 5'exo)(Enzymatics), Klenow Fragment(Enzymatics), 10Xblue buffer(Enzymatics) , DNA Ligase Buffer 10× (Enzymatics), Phusion DNA polymerase (NEB).

[0078] Reagent I pH is 7.9, and solvent is water, and solute is following final concentration material: 50mM sodium chloride solution (Sinopharm Chemical Reagent Co., Ltd.), 100mM Tris-HCl solution (Sinopharm Chemical Reagent Co., Ltd.), 100mM MgCl 2 (Sinopharm Chemical Reagent Co., Ltd.), 10 mM dithiothreitol (Sigma), 5 mM dATP (Enzymatics), 20 U / ml Klenow Fragment (3′-5′exo).

[0079] The pH of reagent II is 7.6, the solvent is water, and the solute is the following final concentration substances: 72U / ml T4DNA polymerase, 10mM ATP, 500mM Tris-HCl solution, 100mMMgCl 2 , 50mM dithiothreitol;

[0080] 1.2 Experimental steps

[0081] 1.2.1 Take two 50ng / μl Fosmid samples (Shenzhen Huada...

Embodiment 2

[0114] 2.1 Reagents

[0115] 10×blue buffer (Enzymatics), pUC18DNA (Takara).

[0116] Reagent I is the same as that described in Item 1.1 of Example 1.

[0117] Reagent II pH is 7.6, the solvent is water, the solute is T4DNA ligase (Enzymatics), ATP (NEB), 100mM buffered saline solution, 100mM MgCl 2 (Sinopharm Chemical Reagent Co., Ltd.), 50 mM dithiothreitol (Sigma).

[0118] 2.2 Experimental steps

[0119] 2.2.1 Add "A", draw two copies of PCR products (200bp) amplified with pUC18DNA as a template (Shenzhen Huada Gene Research Institute) and place them in EP tubes, name them X and Y respectively, and configure the reaction according to the following table system.

[0120]

[0121] Reaction temperature: 37° C., 30 min. Sample Y was purified by a DNA product purification kit (QIAquick PCR Purification Kit from QIAGEN Company) and dissolved in 16.4 μl of EB eluent (column purification will lose 2 μl of liquid volume).

[0122] 2.2.5 Add "Adapter",

[0123]

[0124]...

Embodiment 3

[0131] 3.1 Reagents

[0132] T4 DNA ligase (Rapid, L603-HC-L) (Enzymatics).

[0133] Reagent I is the same as that described in Item 1.1 of Example 1.

[0134] Reagent II pH 7.6, solvent is water, solute is T4DNA ligase (Takara), 10mM ATP (NEB), 500mM Tris-HCl solution, 100mM MgCl 2 (Sinopharm Chemical Reagent Co., Ltd.), 50 mM dithiothreitol (Sigma).

[0135] 3.2 Experimental steps [Agilent Technologies.Sureselect target enrichment system for illumina paired-end sequencing library.G3360-90020]

[0136] 3.2.1 Take two 50ng / μl Fosmid samples (Shenzhen Huada Gene Research Institute) and place 40μl each in Covaris sample tubes, named X and Y respectively, and use the E210 model breaker produced by Covaris to break the samples. The interrupt parameters are as follows:

[0137]

[0138] 3.2.2 For details on the end repair operation, refer to the Agilent SureSelect platform operation manual G3360-90020.

[0139] 3.2.2 Add "A", and process samples X and Y according to the rea...

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Abstract

The invention provides a novel library construction method based on an illumina sequencing platform, and in particular provides a small-fragment DNA library construction method. Aiming at the defects of the small-fragment DNA library construction of the existing illumina sequencing platform, the purification steps can be reduced, the loss and waste of a library are reduced, the cost is lowered, and the working efficiency is improved.

Description

technical field [0001] The invention relates to genome sequencing, especially the field of high-throughput genome sequencing. More particularly, the method of the present invention relates to a library construction method based on the illumina sequencing platform, especially a small fragment library construction method. Background technique [0002] Deoxyribonucleic acid (DNA) is a molecular compound of a double helix structure formed by two nucleotide chains based on the principle of complementary pairing. It is the carrier of life genetic information. Sequencing analysis of DNA nucleotide sequences not only provides important data for basic biological research such as gene expression and gene regulation, but also plays an important role in applied research fields such as disease diagnostics and gene therapy [Gilbert W. DNA sequencing and gene structure.In: Forsén S.Nobel Lectures in Chemistry 1971-1980.1980.Singapore:World Scientific Publishing Co, 1993, 408-426.Sanger F,...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08C12Q1/68
CPCC40B50/06C40B40/08C12Q1/68C12Q1/6806
Inventor 栾合密张俊青程玲孔淑娟张秀清杨焕明
Owner WUXI QINGLAN BIOLOGICAL SCI & TECH
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