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Kit, database establishment method, and method and system for detecting area target variation

A target region and kit technology, applied in the field of biomedicine, can solve the problem of inability to distinguish low-frequency mutations in tumor specimens with sequencing errors, and achieve low false positive rate, high cost performance, pertinence, and high specificity

Inactive Publication Date: 2016-09-07
GUANGZHOU JINGKE DX CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Nowadays, high-throughput sequencing technology has been widely used in medical research. However, due to the low content of free plasma DNA in the early stage of lung cancer, and the sequencing technology itself has a certain error rate, traditional sequencing methods will not be able to distinguish sequencing errors. and low-frequency mutations in tumor samples, so the development of an easy-to-operate, low-injury, and high-accuracy technology is a difficult point in the field of early detection of lung cancer.

Method used

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  • Kit, database establishment method, and method and system for detecting area target variation
  • Kit, database establishment method, and method and system for detecting area target variation
  • Kit, database establishment method, and method and system for detecting area target variation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment one design chip

[0070] 1. Count the number of mutation samples in each exon region of the driver gene related to lung cancer caused by a single gene in the OMIM data and related literature, the number of mutation samples, the number of samples where the hottest mutation is located, and the PI value (to evaluate the frequency of patient response in The level of each exon, PI=the cumulative number of patients carrying mutations in each exon / exon length), and arranged in descending order according to the PI value. Then, take the sample of the first exon region mutation as the sample database, count the number of different samples in all other intervals and sample databases, and list the sample interval with the largest number of different samples as the second screening interval to the chip. At the same time, the mutation samples of the two intervals screened are used as the sample database, and the third interval is screened in the same way until the sample d...

Embodiment 2

[0075] Example 2 Construction of the target region sequencing library, the specific process see figure 1 .

[0076] (1) Sample preparation

[0077] 1. Take 5-10mL of peripheral blood from the subject, store it in an EDTA anticoagulant tube, and separate the peripheral blood within 4-6 hours;

[0078] 2. Plasma cell-free DNA extraction (refer to the QIAamp Circulating Nucleic Acid Kit extraction reagent manual for plasma cell-free DNA extraction); obtain plasma cell-free DNA (cfDNA), which may contain DNA fragments (ctDNA) from tumor cells. (2) Library construction

[0079] 1. End repair

[0080]

[0081] After the reaction, add 120 μL of Agencourt AMPure XPreagent, after the magnetic beads are purified, finally redissolve 42 μL ddH2O, and carry out the next reaction with magnetic beads;

[0082] 2. Add A at the end

[0083]

[0084] After the reaction, add 90 μL of PEG / NaCl SPRI solution, mix well and perform magnetic bead purification, and finally redissolve (35-li...

Embodiment 3

[0098] Example 3 On-machine sequencing

[0099] The sequencing library obtained in Example 2 was sequenced on the computer using the Nextseq CN 500PE75 program, and the sequencing experiment was performed in accordance with the operating instructions provided by the manufacturer (see the cBot officially announced by Hangzhou Berry Hekang Gene Diagnostics Co., Ltd.).

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Abstract

The invention provides a kit comprising a probe. The probe is fixed on a solid substrate or is free in a solution; the probe can specifically recognize a target area, wherein the target areas include one of the followings: at least one of the147 genes shown in Table 1; or a CDS area of at least one gene in Table 1; or the upstream and downstream 10-200bp area of a CDS area of at least one gene in Table 1. The invention also provides the application of the kit, a method for constructing a target area sequencing library, a sequencing method, and a method and a system for detecting the variation of the target area. The kit and / or method and system can simply and conveniently obtain lung cancer-related gene sequences at one time and high specificity, and accurately detect and analyze these related gene sequences, so that the detection and analysis results can assist the study of lung cancer.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, to a kit and its use, more specifically, the present invention relates to a kit, the use of the kit, a method for constructing a sequencing library of a target region, a sequencing method and a A method and system for detecting variation in a target region. Background technique [0002] Primary lung cancer (hereinafter referred to as lung cancer) is one of the most common malignant tumors in my country. According to the data released by the National Cancer Registry Center in 2014, in 2010, there were 605,900 new cases of lung cancer in my country (416,300 males and 189,600 females), ranking first in malignant tumors (first in males and second in females), accounting for 605,900 new cases of malignant tumors. 19.59% of cases (male 23.03%, female 14.75%). The incidence rate of lung cancer was 35.23 / 100,000 (male 49.27 / 100,000, female 21.66 / 100,000). During the same period, the num...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/00C12M1/34C40B50/06
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2525/191C12Q2531/113
Inventor 韩颖鑫张印新王佳伟高晓峘张春生李胜
Owner GUANGZHOU JINGKE DX CO LTD
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