Method and kit for quickly building plasma DNA sequencing library

A DNA sequencing and kit technology, which is applied in the field of plasma DNA library construction for high-throughput sequencing, can solve the problems of high molecular biology operation ability, inaccurate determination results, uneven coverage, etc. Base bias and heterogeneity of genome coverage and inaccuracy of sequencing results, the authenticity of plasma DNA sequencing results, and the effect of reducing sample confusion and contamination

Active Publication Date: 2016-02-03
北京贝瑞和康医学检验实验室有限公司
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AI Technical Summary

Problems solved by technology

The construction method of this plasma DNA sequencing library requires 6 main enzymes, 4 enzyme reaction systems, and 4 times of cleaning and purification, so the cost is high, the operation is complicated, the environment is harsh, and it is easy to form aerosol pollution. At the same time, the requirements for the operator's ability to operate molecular biology are very high, and it is difficult to realize the simultaneous processing of multiple samples.
In addition, the aerosol pollution caused by the PCR process can easily cause the contamination of the sequencing sample. At the same time, the PCR process will also introduce a certain base preference, so that the coverage of the sequencing results on the genome will also be uneven, resulting in uneven judgment results. accuracy

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  • Method and kit for quickly building plasma DNA sequencing library
  • Method and kit for quickly building plasma DNA sequencing library
  • Method and kit for quickly building plasma DNA sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction method of the plasma DNA sequencing library of embodiment 1 mainly includes the following steps (please refer to Figure 4 ):

[0054] (1) Extraction of plasma DNA: This step can be carried out using any methods and reagents suitable for extracting plasma DNA well known to those skilled in the art.

[0055] (2) End filling, followed by cleaning and purification of the product: this step can be performed using any methods and reagents known to those skilled in the art that are suitable for end filling and subsequent cleaning and purification. For example, T4 DNA polymerase, Klenow enzyme can be used as an enzyme for end filling.

[0056] (3) Overhang A at the end, followed by cleaning and purification of the product: This step can be performed using any methods and reagents known to those skilled in the art that are suitable for overhang A at the end and subsequent cleaning and purification of the product. For example, under the action of klenowex-(New...

Embodiment 2

[0083] The construction method of the plasma DNA sequencing library in Example 2 is basically similar to Example 1, the difference is that in Example 2, the step of filling in the end is omitted, and the extracted plasma DNA is directly suspended at the end and purified. (please refer to Figure 4 ).

[0084] A specific example of plasma DNA sample sequencing using the method according to Embodiment 2 of the present invention is shown below.

[0085] Step 1: Extract about 5ng of plasma DNA.

[0086] Step 2: Prepare the reaction mixture shown in Table 4, and incubate at 37°C for 30 minutes to add a polyadenine tail to the 3' end of the DNA fragment; purify the DNA sample on a column, and incubate in 37 μl of sterile dHO 2 O or elution buffer.

[0087] Table 4

[0088]

[0089]

[0090] Step 3: Prepare the reaction mixture for DNA fragment ligation and sequencing adapters shown in Table 5, incubate at 20°C for 15 minutes, incubate at 65°C for 10 minutes, and keep at 4°...

Embodiment 3

[0097] The construction method of the plasma DNA sequencing library in Example 3 is basically similar to Example 1, the difference is that in Example 3, the end filling and the end hanging A are carried out in one reaction system, and the end filling and the end hanging A are carried out in one reaction system. There is no cleaning and purification step in between, and the common Taq enzyme is used instead of the commonly used klenowex-enzyme to make the buffer system of the two reactions compatible (please refer to Figure 4 ).

[0098] A specific example of plasma DNA sample sequencing using the method according to Embodiment 3 of the present invention is shown below.

[0099] Step 1: Extract about 5ng of plasma DNA.

[0100] Step 2: Prepare the reaction mixture shown in Table 6, incubate at 37°C for 20 minutes (end filling), and incubate at 72°C for 20 minutes (end hanging A), so as to perform end filling and end in one reaction system Suspension A; DNA samples were purif...

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Abstract

The invention provides a method for quickly building a plasma DNA sequencing library. The method includes the following steps: 1), extracting plasma DNA; 2), performing end filling on the plasma DNA to obtain plasma DNA subjected to end filling; 3), performing 3' suspension A on the plasma DNA subjected to end filling to obtain plasma DNA after 3' suspension A; 4), connecting the plasma DNA after 3' suspension A with a sequencing joint to obtain a connection product; 5), purifying the connection product to obtain a purified product. The invention further provides a kit for building the plasma DNA sequencing library. Building process of the plasma DNA sequencing library is simplified greatly, pollution of environment and pollution among samples due to PCR amplification is removed, requirements, on environment, of building of the plasma DNA library are lowered, nonuniformity in genome covering caused by the process of PCR is avoided, building of the plasma sample library is enabled to be lower in cost, higher in efficiency and quick, and plasma DNA sequencing results are truer and more effective.

Description

technical field [0001] The present invention relates to a method and a kit for constructing a plasma DNA sequencing library, more particularly, the present invention relates to a method and a kit for constructing a plasma DNA library for high-throughput sequencing. Background technique [0002] With the advancement of science and technology, traditional Sanger sequencing can no longer fully meet the needs of research. Genome sequencing requires lower cost, higher throughput, and faster sequencing technology. High-throughput sequencing (also known as second-generation sequencing) ) technology came into being. The core idea of ​​high-throughput sequencing technology is sequencing-by-synthesis, that is, to determine the DNA sequence by capturing the markers of newly synthesized ends. The existing technology platforms mainly include Roche / 454FLX, Illumina / Hiseq, Miseq, NextSeq and LifeTechnologies / SOLIDsystem, PGM, Proton, etc. So far, each run of HiSeq2000 can achieve the se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10C40B50/06C40B40/06
CPCC12Q1/6806C12Q2521/131C12Q2525/191C12Q2535/122
Inventor 陈迪李亚丽李天成石燕滨张江花吴凯
Owner 北京贝瑞和康医学检验实验室有限公司
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