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Construction method for lung cancer multi-gene mutation sequencing library and kit

A kit and gene technology, applied in the field of biomedicine, can solve problems such as uniformity decline, quality and storage time impact, and difficult to detect sites, and achieve the effects of reducing sample waste, good uniformity and sensitivity

Pending Publication Date: 2021-04-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are several similar products for lung cancer driver gene detection based on the NGS platform in China. However, these products detect relatively few mutation types and loci, and the coverage, capture rate and uniformity of multiple genes need to be improved, especially those without The testing range of products covers the gene fusion of NTRK1-3, the target of FDA's new anticancer drug larotrectinib
[0005] At present, the library construction methods used for targeted sequencing on the market are mostly multiplex PCR methods. As the number of detection sites increases, the number of primers required increases. Due to the competition of each PCR reaction, the uniformity decreases, and even some sites are difficult to detect. out
And for gene fusion, the multiplex PCR method needs to use mRNA for library construction, and the quality and storage time of RNA also have an impact on the quality of the library; in addition, the library constructed by the multiplex PCR method cannot detect structural variations such as gene amplification / deletion

Method used

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  • Construction method for lung cancer multi-gene mutation sequencing library and kit
  • Construction method for lung cancer multi-gene mutation sequencing library and kit
  • Construction method for lung cancer multi-gene mutation sequencing library and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] A kit for detecting multi-gene somatic mutation and gene fusion in lung cancer, comprising a capture probe mixture, an adapter mixture, and an end repair and tailing reaction system. The capture probe sequences are as follows:

[0083] Table 1 Capture probe sequences

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] The adapter mixture combination is characterized in that different adapter mixtures are used for library construction from different DNA sample sources, and the i5 / i7index sequences of P5 / P7 adapters in each adapter mixture are different, and the combination of i5 / i7index sequences is unique. In each linker mixture, the working concentration of each linker is 150 μM. For library construction of different samples, different adapter mixes should be used.

[0090] The end repair and tail addition reaction system includes end repair and tail addition enzyme, and its components include: 5U T4 DNA ligase, 10U T4 polynucleotide kinase and 5U Ta...

Embodiment 2

[0096] Sample detection was performed using the kit described in Example 1.

[0097] 1. Sample DNA fragmentation

[0098] In this example, using Covaris TM The DNA ultrasonic breaker fragments the DNA sample to 150-400bp, and the enzyme digestion method can also be selected for DNA fragmentation.

[0099] 2. End repair and tailing:

[0100] The reaction system is configured in proportion as shown in Table 3.

[0101] Table 3 End repair and tailing reaction system

[0102] Reagent Dosage Fragmented DNA 50μl End Repair Tailing Buffer 8μl end repair tailing enzyme 2μl Total 60μl

[0103] Mix well and centrifuge briefly, and react according to the procedure in Table 4.

[0104] Table 4 Reaction procedures for end repair and tailing

[0105] temperature time 20℃ 30min 70℃ 20min 4℃ ∞

[0106] 3. Joint connection

[0107] Configure the reaction system, as shown in Table 5.

[0108] Table 5 Adapter ...

Embodiment 3

[0203] Example 3 Influence of DNA samples from different sources on library sequencing results

[0204] Select 30 DNA samples from paraffin tissue sections, fresh tissues or plasma, including 20 samples with positive gene variation and 10 samples with negative gene variation within the detection range verified by Sanger sequencing / FISH method / digital PCR method , use the kit of Example 1, carry out library construction according to the method of Example 2, and perform sequencing analysis, the results show (see Table 17): using the method of the present invention to carry out library construction and sequencing of DNA samples from different sources, the The qualitative results are completely consistent with the gold standard test results such as Sanger sequencing or FISH, and are stable and reliable.

[0205] Table 17 Sequencing results of libraries constructed from DNA samples from different sources

[0206]

[0207]

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PUM

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Abstract

The invention provides a capture probe for amplifying lung cancer driving genes. The capture probe can cover hot spot somatic cell mutation, insertion and deletion, structural variation and gene fusion types of a plurality of lung cancer driving genes such as EGFR, ALK, ROS1, KRAS, BRAF, PIK3CA, HER2, RET, MET, NRAS, NTRK1-3, MAP2K1 and the like; and aiming at NTRK1-3 gene fusion, a second-generation sequencing library does not exist in China at present to cover the fusion type. According to the probe and the prepared kit, compared with a traditional PCR method, the library construction method used for targeted sequencing is a hybrid capture method, various variation types such as hot spot somatic cell mutation, insertion and deletion, structural variation and gene fusion can be effectively detected, and the probe and the kit have good uniformity and sensitivity for detection of multiple genes and multiple sites. Moreover, only DNA samples need to be used in library construction by the hybrid capture method, and extra RNA samples do not need to be extracted for gene fusion detection, so that sample waste is reduced, and the influence of RNA quality on library construction does not need to be considered.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method and a kit for constructing a lung cancer multi-gene mutation sequencing library. Background technique [0002] There are about 781,000 cases of lung cancer in my country each year, and about 626,000 cases of death. It is currently the cancer with the highest morbidity and mortality in my country. Based on histopathological results, lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which non-small cell lung cancer accounts for about 80%. In recent years, with the continuous development of molecular diagnostic techniques and the emergence of new targeted drugs, the treatment of non-small cell lung cancer has entered the era of targeted therapy. [0003] Non-small cell lung cancer driver genes include EGFR, ALK, ROS1, KRAS, BRAF, PIK3CA, HER2, RET, MET, NRAS, NTRK1-3, MAP2K1 and other genes, which are relat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11C40B50/06
CPCC12Q1/6858C40B50/06C12Q2535/122C12Q2531/113
Inventor 曾杰彭璨璨吴诗扬刘志明
Owner SUREXAM BIO TECH