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Construction method, kit and application of plasma free DNA methylation detection library

A construction method and methylation technology, which is applied in the field of plasma cell-free DNA methylation detection library construction, can solve the problems that plasma cell-free DNA cannot construct a library, reduce the initial amount of library construction, improve library recovery efficiency, simplify The effect of the library building step

Inactive Publication Date: 2018-01-05
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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Problems solved by technology

[0009] The present invention aims to provide a method for constructing a plasma free DNA methylation detection library, a kit and its application to solve the technical problem in the prior art that the plasma free DNA cannot be constructed using the conventional micro-methylation library construction method

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  • Construction method, kit and application of plasma free DNA methylation detection library
  • Construction method, kit and application of plasma free DNA methylation detection library
  • Construction method, kit and application of plasma free DNA methylation detection library

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Embodiment approach

[0028] According to a typical embodiment of the present invention, a method for constructing a plasma cell-free DNA methylation detection library is provided. The construction method includes the following steps: S1, extracting plasma free DNA from blood samples; S2, performing end repair on plasma free DNA and adding A base to the 3' end; S3, connecting the end of plasma free DNA obtained in S2 to C Modified linker (design principle: phosphorylation at the 5' end of the F chain, all C methylation modifications, all C methylation modifications on the R chain); S4, perform bisulfite conversion on the product obtained from S3, and perform PCR extension; S5, perform magnetic bead purification on the PCR product of S4, remove small fragments of DNA and primer dimers that have not been non-specifically amplified; and S6, perform PCR amplification on the product of S5, and perform magnetic bead purification, Obtain plasma cell-free DNA methylation detection library.

[0029] Prefer...

Embodiment 1

[0045] The main experimental steps are as follows:

[0046] 1. Acquisition of plasma samples

[0047] Under normal laboratory conditions, use EDTA blood collection tubes, preferably Streck blood collection tubes to collect 10ml of peripheral blood from prostate cancer patients, centrifuge quickly at room temperature for 10min at 1900g (or 3000rpm), and gently draw the upper layer of plasma (to avoid buffy coat contamination) into a new 15ml centrifuge tube , centrifuge at 16000g room temperature for 10min, and gently pipette the supernatant into a new 15ml centrifuge tube.

[0048] 2. Extraction and quality inspection of plasma cell-free DNA

[0049] Use Qiagen’s kit for extracting free nucleic acid under routine laboratory conditions, preferably Circulating Nucleic Acid kit, extract free DNA from the separated plasma according to the instructions, and elute with 30 μl warm water, and use Agilent 2100 to qualitatively and quantitatively detect the extracted plasma free DNA,...

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Abstract

The invention discloses a construction method, a kit and application of a plasma free DNA methylation detection library. The construction method comprises the following steps: S1 of extracting plasmafree DNA from a blood sample; S2 of performing tail end repair and 3' terminal A base addition on the plasma free DNA; S3 of connecting the tail ends of the plasma free DNA obtained in the S2 with C methylation modified connectors; S4 of performing bisulfite conversion on a product obtained in S3 and performing PCR expansion; S5 of performing magnetic bead purification on the PCR product of S4 andremoving small-fragment DNA which is not amplified in a non-specific mode and primer dimer; S6 of performing PCR amplification on a product of S5 and performing magnetic bead purification to obtain aplasma free DNA methylation detection library. By means of the technical scheme of the construction method disclosed by the invention, an initial amount of general microscale methylation library construction is reduced, library construction steps are simplified, library recycling efficiency is improved, and a foundation is established for sequentially screening tumor diagnosis markers through plasma free DNA methylation difference locus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a plasma free DNA methylation detection library, a kit and an application thereof. Background technique [0002] Plasma free circulating tumor DNA (circulating tumor DNA, ctDNA) refers to single-stranded or double-stranded DNA released into plasma by necrotic or apoptotic tumor cells, accounting for about 0.1%-1% of free DNA. The size of the ctDNA fragment is usually 160-180bp, with a half-life of 2 hours in the blood, and carries information such as SNP (point mutation), InDel (insertion deletion), CNV (copy number), fusion (fusion gene), methylation, etc. ctDNA flows continuously in the blood circulation system, which can reflect the current information of tumor patients in real time. [0003] Compared with traditional detection methods, ctDNA detection has many advantages: 1) Convenient sampling: ctDNA detection only needs to draw blood to complete the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/68
Inventor 孙英丽任善成常双
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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