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DNA library kit for sequencing and construction method of DNA library for sequencing

A library construction and kit technology, applied in DNA/RNA fragments, recombinant DNA technology, libraries, etc., can solve the problems of time-consuming, high consumption of purification reagents, and high cost of the overall library construction process, and achieve high ligation efficiency and effective library. High yield and control of nucleic acid contamination

Inactive Publication Date: 2019-02-12
FAPON BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And after the end of each process, the DNA needs to be purified before proceeding to the next step. The whole process takes a long time, consumes a lot of purification reagents, and the cost of the overall library construction process is relatively high.

Method used

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  • DNA library kit for sequencing and construction method of DNA library for sequencing
  • DNA library kit for sequencing and construction method of DNA library for sequencing
  • DNA library kit for sequencing and construction method of DNA library for sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The library construction method based on the Illumina sequencing platform, the steps are as follows:

[0093] (1) Sample preparation: Escherichia coli (E.coli) genome was extracted from E. coli LB medium, and the extraction method was Omega whole genome extraction kit. The extracted large fragments of DNA need to be physically interrupted by Covaris, and the interrupted DNA sequence is a small fragment of DNA of about 200bp. The fragmented DNA fragments were purified by Ampure XP magnetic beads, diluted to a concentration of 10 ng / μl with TE buffer, and OD260 / OD280=2.0.

[0094] (2) Fragmented DNA (DNA Fragment) end repair and A: Dilute the fragmented DNA interrupted by step (1) to an appropriate concentration, use end repair and add A reagents for end repair and insert into the fragmented DNA An adenosine (A base) is added to the 3' end of the sequence.

[0095] The reaction system is shown in Table 1.

[0096] Table 1 reaction system

[0097]

[0098] Among the...

Embodiment 2

[0136] The difference from Example 1 is that the end repair and the components of A buffer: Tris-HCl 10mM, ATP 1mM, MgCl 2 10mM, dNTP 100μM, dATP 1mM;

[0137] End repair and A enzyme use 5U of Bst DNA polymerase;

[0138] End repair with plus A: incubate at 20°C for 30 minutes, and at 37°C for 30 minutes.

[0139] Components of ligation reaction buffer: Tris-HCl 10mM, ATP 1mM, MgCl 2 10mM, 2% PEG4000;

[0140] The ligase is a mixture of equal volumes of 50 U of T4 DNA ligase, 1 U of Bst DNA polymerase, and 1 U of T4 polynucleotide kinase.

[0141] The ligation condition is: incubate at 20°C for 30 minutes.

[0142] The rest are the same as in Example 1, and the effect of building a library is basically the same as in Example 1.

Embodiment 3

[0144] The difference from Example 1 is that the end repair and the components of A buffer: Tris-HCl 50mM, ATP 3mM, MgCl 2 50mM, dNTP 300μM, dATP 5mM;

[0145] End repair and A-adding enzyme use 5U of Klenow enzyme (3'-5'EXO-) and 5U of T4 DNA polymerase in a volume ratio of 1:1 mixed enzyme;

[0146] End-repair with Add A: Incubate at 30°C for 15 minutes and at 65°C for 15 minutes.

[0147] Components of ligation reaction buffer: Tris-HCl 50mM, ATP 3mM, MgCl 2 50mM, 8% PEG6000;

[0148] The ligase is T4 DNA ligase with 200 U of ligase.

[0149] The ligation conditions were: incubate at 37°C for 15 minutes.

[0150] The rest are the same as in Example 1, and the effect of building a library is basically the same as in Example 1.

[0151] In addition, in the present invention, the length of the linker sequence can be other commercially available linkers suitable for the Illumina platform, or it can be the 20-50 bp base sequence of the linking part of the linker and the i...

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Abstract

The invention relates to the field of gene sequencing, and in particular to a DNA library kit for sequencing and a construction method of a DNA library for sequencing. The construction method of the DNA library for sequencing comprises the following steps: DNA fragmentation is preformed for terminal repair and addition of A, an obtained product is ligated to a linker and purified, the DNA fragmentis subjected to size screening; amplification and enrichment, an obtained product is purified to obtain the DNA library. The method simplifies the process of DNA library construction, the steps of DNA sequence terminal repair and addition of A at 3' end are completed in one step, and the product can be directly used for ligation reaction without intermediate purification, which greatly shortens the experimental time consumed by the process of library construction; the unique linker design greatly saves the synthesis cost of the linker, which provides great convenience for the experimental staff of the library construction, reduces the consumption of reagents, and reduces the overall library construction cost.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a DNA library kit for sequencing and a method for constructing a DNA library for sequencing. Background technique [0002] High-throughput sequencing technology, called next generation sequencing in some literatures, is an epoch-making revolutionary change to traditional sequencing (Sanger sequencing). The molecular sequence is determined. At the same time, high-throughput sequencing makes it possible to conduct a comprehensive analysis of the transcriptome or genome of a species. NGS technology can be used to sequence the DNA (or RNA) of any organism, providing valuable genetic information. As a highly scalable technology, DNA sequencing can be applied to specific regions of interest or to sequence entire genomes, helping researchers investigate and discover, and better understand health and disease. [0003] Illumina next-generation sequencing (NGS) technology uses a sequencin...

Claims

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Application Information

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IPC IPC(8): C12N15/11C40B50/06C40B40/06
CPCC12N15/11C40B40/06C40B50/06
Inventor 王洋蔡统聪张志毅章瑞程杨倩
Owner FAPON BIOTECH INC
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