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Low-initial-quantity plasma free DNA methylation library-building kit and method

A technology of methylation and kits, which is applied in the field of methylation library construction methods and library construction kits, can solve problems such as difficult to effectively meet the requirements of DNA input volume, high DNA input volume, and high amplification cycle number, and achieve The effect of improving the quality of library construction and sequencing, reducing purification steps, and reducing input requirements

Pending Publication Date: 2020-01-10
广州迈格基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the main technology to detect methylation by next-generation sequencing is to treat genomic DNA with bisulfite (Bisulfite method), and the treatment of DNA with bisulfite will lead to a large loss of DNA, which requires more time for methylation detection. Problems with high DNA input and high amplification cycle numbers
Because the content of cfDNA is very small, it is often difficult to effectively meet the DNA input requirements of the bisulfite-based methylation library construction method, and the excessively high number of amplification cycles will introduce more sequencing errors. Therefore, the traditional cfDNA Methylation libraries are often of low quality

Method used

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  • Low-initial-quantity plasma free DNA methylation library-building kit and method
  • Low-initial-quantity plasma free DNA methylation library-building kit and method
  • Low-initial-quantity plasma free DNA methylation library-building kit and method

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Experimental program
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Effect test

Embodiment 1

[0080] 1. End repair and A addition

[0081] Use the nucleic acid-containing solution as template DNA for end repair and A addition reactions.

[0082] Reagents: ER / dA enzyme mixture (containing T4 polynucleotide kinase, Taq DNA polymerase, T4 DNA polymerase), 5XER / dA reaction buffer (containing dNTP, Tris-HCl, MgCl 2 , DTT).

[0083] The input amount of template DNA in the 50μl reaction system is at least 1ng, the concentration of T4 polynucleotide kinase is 0.6U / μl, the concentration of Taq DNA polymerase is 0.3U / μl, the concentration of T4 DNA polymerase is 0.5U / μl, and the concentration of dATP is 0.15nmol / μl, dNTP concentration is 0.3nmol / μl, Tris-HCl concentration is 80mM, MgCl 2 The concentration was 15 mM and the concentration of DTT was 7 mM.

[0084] Reaction conditions: incubate at 30°C for 30 minutes, then incubate at 72°C for 30 minutes.

[0085] Specific reaction system:

[0086] Reagent Volume μl template DNA X nuclease free water 32....

Embodiment 2

[0133] The present invention designs a DNA ligase enhancement solution, and tests its effect on the methylation library construction of the present invention.

[0134] (1) Test the effect of hexaammine cobalt chloride on the methylation library building of the present invention, taking 10ngDNA input as an example, the results are as follows:

[0135]

[0136] (2) Test the effect of betaine on methylation library construction of the present invention, taking 10ngDNA input as an example

[0137]

[0138] The results showed that hexaammine cobalt chloride could improve the quality of the library; further addition of betaine could improve the quality of the library to a certain extent.

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Abstract

The invention discloses an enzymatic methylation library-building method. According to the method, through TET enzyme methylation treatment, DNA damage in the methylation library-building process is lowered to a certain extent, and thus the requirement for the input amount of DNA in a nucleic acid sample is lowered; besides, by further cooperating with a one-step reagent for terminal repair and Aaddition reaction, one-tube library building can be realized, and the purification step is reduced; and by further cooperating DNA ligase enhancing liquid, the connection efficiency can be improved, and the library-building quality and the sequencing quality are improved; and the method provided by the invention is low in library-building initial quantity, the DNA input amount can be as low as to1 ng, and the method is suitable for trace library building including cfDNA.

Description

technical field [0001] The invention belongs to the field of methylation sequencing, and more specifically relates to a method for building a methylation library and a kit for building a library. Background technique [0002] Blood free DNA (cell-free DNA, cfDNA) is a DNA fragment discharged into the blood by human tissues. In healthy people, a small amount of free DNA can be found in plasma, and the fragments are mainly 160-175bp or integer multiples. Cell-free DNA in healthy human plasma mainly comes from apoptotic cells. Compared with healthy people, the content of cfDNA in tumor patients is increased, and these cfDNA can come from tumor tissues, circulating tumor cells and normal tissues. ctDNA (circulating tumor DNA) is a kind of all free circulating DNA (cfDNA) in body fluids, and it is a free genome fragment carrying tumor-specific genetic changes released only by tumor cells. ctDNA can reveal the comprehensive genetic information of tumors, accurately reflect the h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12Q1/6806C12N15/1093C40B50/06C12Q2525/191C12Q2521/539C12Q2531/113
Inventor 夏渝东郑子鹏沈俊芳
Owner 广州迈格基因科技有限公司
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