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A method for constructing a cell-free DNA library and a method for detecting low-frequency mutations in cell-free DNA

A DNA library and construction method technology, applied in the detection of low-frequency mutations in free DNA, and in the field of free DNA library construction, can solve the problems of complex detection methods and high costs, and achieve the effect of overcoming sequencing errors and amplification errors and retaining mutation frequencies

Active Publication Date: 2019-03-19
元码基因科技(苏州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires secondary amplification, and the detection method is complicated and costly

Method used

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  • A method for constructing a cell-free DNA library and a method for detecting low-frequency mutations in cell-free DNA
  • A method for constructing a cell-free DNA library and a method for detecting low-frequency mutations in cell-free DNA
  • A method for constructing a cell-free DNA library and a method for detecting low-frequency mutations in cell-free DNA

Examples

Experimental program
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Embodiment 1

[0070] Example 1 Detection method of cfDNA low-frequency mutation in plasma

[0071] 1. Sample extraction

[0072] Use the free DNA extraction reagent to extract the free DNA in the subject's plasma. The main steps are as follows: temporarily store the whole blood sample in ice at 4°C, turn on the high-speed refrigerated centrifuge, put the blood collection tube at 4°C and centrifuge at 1600g for 10 minutes to remove residual cells, and then centrifuge at 16000g at 4°C After 10 minutes, remove residual cells and transfer the supernatant to a new 2 mL centrifuge tube.

[0073] Add 600 μL of proteinase K to a 2 mL centrifuge tube to fully dissolve and mix the plasma, vortex to mix for 10 seconds, and centrifuge briefly.

[0074] Add 600 μL of the mixed buffer solution GB mix, briefly centrifuge; place in a water bath at 56°C for 10 minutes; take out the centrifuge tube, cool at room temperature for 5 minutes, and briefly centrifuge; add 300 μL of frozen absolute ethanol, gentl...

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Abstract

The invention relates to a free DNA library construction method. A 3' end of a single-stranded free DNA is connected with a sole sequence tag; the sole sequence tag comprises a random sequence and a linker sequence; an upstream primer and a downstream primer for PCR amplification are added for PCR amplification; the upstream primer is a primer designed aiming at a target low frequency mutation; the downstream primer is the primer corresponding to the linker sequence. In addition, the invention further relates to a detection method for low and medium frequency mutation in a free DNA. The 3' end of the single-stranded free DNA is connected with the sole sequence tag; the upstream primer and the downstream primer for PCR amplification are added for PCR amplification; one end of each of the upstream primer and the downstream primer is connected with the sequence tag respectively; the amplified DNA is sequenced online on an Illumina platform. According to the method, detection on cfDNA low frequency mutation can be simply and efficiently achieved; the target of fetal inheritance or tumor cell mutation detection can be achieved through cfDNA detection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a free DNA library and a method for detecting low-frequency mutations in the free DNA. Background technique [0002] Free DNA (Cell Free DNA, referred to as cfDNA) refers to the free DNA in peripheral blood, which exists in the form of naked nucleic acid, and mainly comes from the fragmented DNA produced in the process of cell apoptosis or cell lysis (such as caused by physical stimulation). DNA fragments produced after necrosis or immune cell killing), the size is basically around 160-180bp. [0003] As early as 1947, Mandel et al. found free DNA in the serum and plasma of healthy people, but it did not attract enough attention at that time. In 1977, Leon et al. found that there was a certain relationship between tumor and free DNA content in serum. Afterwards, researchers detected oncogene mutations in the plasma and serum of tumor patients, which wer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869C12Q1/6883C12Q1/6886
CPCC12Q1/6869C12Q1/6883C12Q1/6886C12Q2600/156C40B50/06C12Q2535/122C12Q2525/191C12Q2525/179
Inventor 田埂
Owner 元码基因科技(苏州)有限公司