Construction method of ctDNA ultra-low-frequency mutation detection library, kit and analysis method of library detection data

A mutation detection and construction method technology, applied in the biological field, can solve the problems of decreased sensitivity, limited number of DNA molecules, and low total amount of free DNA, so as to achieve the effect of improving specificity and unaffected sensitivity

Inactive Publication Date: 2017-06-13
天津诺禾医学检验所有限公司
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Problems solved by technology

2) The total amount of free DNA in the blood is very low. On average, only about 50ng of free DNA can be extracted per 10ml of whole blood, and there are many types of tumor-related mutations. Conventional techniques cannot use such a small amount...

Method used

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  • Construction method of ctDNA ultra-low-frequency mutation detection library, kit and analysis method of library detection data
  • Construction method of ctDNA ultra-low-frequency mutation detection library, kit and analysis method of library detection data
  • Construction method of ctDNA ultra-low-frequency mutation detection library, kit and analysis method of library detection data

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Embodiment 1

[0039] 1. 10ml of whole blood from patients with non-small cell lung cancer is collected and transported in BCT tubes from Streck Company. The transport temperature is room temperature and the transport time does not exceed 72 hours. Plasma was separated by two-step centrifugation, that is, centrifuged at 1600g for 10 minutes, the supernatant was taken, and then centrifuged at 16000g for 10 minutes. The supernatant was the separated plasma, which was stored at -80°C. The cfDNA in plasma was extracted using Qiagen’s circulating DNA extraction kit, and the extracted cfDNA was stored at -20°C for later use. See Table 1 for sample names and extraction volumes.

[0040] Table 1

[0041]

[0042] 2. Synthesize the linker with the tag sequence.

[0043] The linker sequence is shown in Table 2 (the length of the tag sequence used in this example is 12 bases), and a maximum of 4 12 a DNA molecule. The synthesized primers were dissolved in EB (Elution Buffer) elution buffer, with...

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Abstract

The invention discloses a construction method of a ctDNA ultra-low-frequency mutation detection library, a kit and an analysis method of library detection data. The construction method comprises steps as follows: S1, cfDNA is extracted from whole blood; S2, the terminal of cfDNA is restored and an A basic group is added to the 3'terminal; S3, the terminal of the cfDNA obtained in S2 is connected with connectors containing random label sequences; S4, primers for multiplex PCR (polymerase chain reaction) are designed according to the sequences of the connectors and an object region for object region capturing; S5, a PCR product in S4 is subjected to magnetic bead purification, and small-fragment DNA not subjected to non-specific amplification and primer dimer are removed; S6, a product in S5 is subjected to PCR amplification, an index sequence is introduced, and the ctDNA ultra-low-frequency mutation library is obtained. According to the method, PCR mistakes and sequencing mistakes are eliminated, and the detection specificity is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a ctDNA ultra-low frequency mutation detection library, a kit and an analysis method for library detection data. Background technique [0002] ctDNA (circulating tumor DNA), that is, circulating tumor DNA, is the DNA released by tumor cells into the blood circulation system. They carry all the gene mutation information in tumor cells. Therefore, in theory, it can be reflected by detecting the mutation status of ctDNA Mutation status of tumor tissue. Because no matter the tumor at the primary site or the tumor at the metastatic site, tumor DNA is continuously released into the blood, so ctDNA reflects the overall tumor gene mutation status of the patient, so it is more comprehensive; only one tube of blood can be extracted for The convenience of detection also enables it to overcome the problems of difficult sampling of tumor tissue and inconvenient long-ter...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C12Q1/68G06F19/28
CPCC12N15/1093C12Q1/6806C40B50/06G16B50/00C12Q2525/191C12Q2531/113C12Q2537/143C12Q2563/143C12Q2563/149
Inventor 朱嘉麒张振宇段楠蒋智
Owner 天津诺禾医学检验所有限公司
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