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Method for constructing a nucleic acid library, reagent and kit

A fragment and labeling technology, applied in the field of nucleic acid library establishment, can solve problems such as incompatibility of sequencing primers, and achieve the effect of improving throughput

Inactive Publication Date: 2012-10-03
WUJIANG HUIJIE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it requires sequencing primers that are not compatible with those of existing sequencing platforms, a point that has disappointed many potential users
[0003] Another challenge facing the field of second-generation sequencing library construction is that the sequencing platform is developing very fast. The development of new sequencers only takes a few years, and the replacement is very fast.
The third-generation sequencing technology may also challenge or replace the second-generation technology in the near future

Method used

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  • Method for constructing a nucleic acid library, reagent and kit
  • Method for constructing a nucleic acid library, reagent and kit
  • Method for constructing a nucleic acid library, reagent and kit

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, the construction of the DNA library with terminal labeling

[0022]Use transposase and transposon end sequence (TE) to randomly interrupt the target DNA to make it reach the desired fragment size. Usually, we incubate target DNA, transposase and its specific transposition end sequence in the buffer of transposition reaction. The amount (0.01ng / ul-lug / ul) of target DNA (such as human genomic DNA), transposase (EZ-Tn5 Transposase, EPICENTRE) and its specific transposable end sequence added varies according to different applications. The composition and concentration of the buffer (1×GPSBuffer, 25mM Tris-HCl, 2mM DTT), the temperature and time of incubation also depend on the size of the fragment to be cut. Such as figure 1 Shown: In two different buffer systems, the molecular weight intervals where DNA is broken are different. The reaction can be terminated after adding reaction termination solution (10% sucrose, 66mM EDTA, 20mM TRIS, 0.1% SDS, 0.9% Orang...

Embodiment 2

[0029] Genomic DNA fragments of maize are used as materials, and the target DNA is randomly interrupted by transposase and transposon end sequence (TE) to make it reach the desired fragment size. The buffer system used in the material is (1×GPS Buffer, 25mM Tris-HCl), the samples were processed under different incubation conditions (37°C, 20 minutes), and then the reaction termination solution (10% sucrose, 66mM EDTA, 20mM TRIS , 0.1% SDS, 0.9% Orange G, and 100g / ml proteinase K), the reaction can be terminated after heating at 50°C for 10 minutes. The size of the cleaved DNA fragments can be observed under 1% gel electrophoresis.

[0030] Once the transposition reaction was complete, the transposon end sequence (TE)-tagged DNA fragments were purified using the ZymoDNAClean and Concentrator kit (Zymo Research Corporation, Irvine, CA).

[0031] The purified transposon end sequence (TE)-tagged DNA fragment and a mixture of single-stranded DNA endonuclease (S1nuclease, Promega) ...

Embodiment 3

[0035] Using the genomic DNA of Streptococcus vibrio and the PCR double-stranded DNA fragments circulating in the interrupted Streptococcus DNA as materials, the target DNA was randomly interrupted by transposase and transposon end sequence (TE) to make it achieve our desired fragment size. The buffer system used by the material is ((1×GPS Buffer, 25mM Tris-HCl), the samples were processed under different incubation conditions (25°C, 2 hours), and then the reaction termination solution (10% sucrose, 66mM EDTA, 20mM TRIS , 0.1% SDS, 0.9% Orange G, and 100g / ml proteinase K), the reaction can be terminated after heating at 50°C for 10 minutes. The size of the cleaved DNA fragment can be observed under 1% gel electrophoresis.

[0036] Once the transposition reaction was complete, the transposon end sequence (TE)-tagged DNA fragments were purified using the ZymoDNAClean and Concentrator kit (Zymo Research Corporation, Irvine, CA).

[0037] The purified transposon end sequence (TE)...

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Abstract

The invention provides a method, reagent and a kit for establishing a nucleic acid library with a mark and uses an in vitro transposition method; the method comprises the following steps: firstly, breaking target DNA at random by transposase, and then, treating the target DNA by nuclease and DNA polymerase to generate a DNA library with a mark; according to the invention, any DNA segment which needs a mark is allowed to be connected, so that the generated DNA segment with the mark can be directly applied to different second generation of sequencing platforms which include but are not limited to those instruments manufactured by companies of Illumina, Life Technologies (ABI), Roche, HelicosBiotechnologies, Pacific Biosciences; similarly, the above-mentioned DNA segment can also be applied to sequencing instruments produced by other manufacturers.

Description

technical field [0001] The invention provides a method, a reagent and a kit for establishing a nucleic acid library. In particular, transposases, nucleases and ligases are used during library construction. Background technique [0002] How to effectively and efficiently use DNA and RNA from various materials to build libraries is a challenge for researchers and operators of next-generation sequencing (NGS). Existing technologies include breaking double-stranded DNA by physical means: ultrasonic method or atomization method, which requires a relatively large amount of starting material. This process is lengthy and difficult to perform in a high-throughput manner. Scientists at NEB recently developed the use of Fragmentase, an enzymatic reaction in a centrifuge tube, which is a good alternative to physical fragmentation, but still requires a large amount of starting material. EPICENTRE has developed Nextera reagents, which enable researchers to complete the library construc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1093C12Q2521/10C12Q2521/501
Inventor 王焱
Owner WUJIANG HUIJIE BIOTECH
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