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Sarcoma fusion gene and/or mutation joint detection primer group and kit

A fusion gene and joint detection technology, applied in the field of molecular biology, can solve the problems of inability to detect carcinogenic fusion genes and point mutations

Active Publication Date: 2020-12-18
苏州科贝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, the above methods can only detect known gene fusions or point mutations, and are limited by the detection throughput, and cannot realize one-time full detection of oncogenic fusion genes and point mutations. Therefore, the development of a A kit that can simultaneously detect fusion genes and point mutations is of great significance for clinical research and diagnosis of soft tissue sarcoma

Method used

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  • Sarcoma fusion gene and/or mutation joint detection primer group and kit
  • Sarcoma fusion gene and/or mutation joint detection primer group and kit
  • Sarcoma fusion gene and/or mutation joint detection primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Design of special adapters for DNA library construction, specific compound primers for detecting fusion genes and point mutations, and amplification compound primers

[0089] 1. Special adapters for DNA library construction

[0090] A special linker for DNA library construction, which is 8 dimers composed of linker primer 1 and 8 different i5-terminal primers respectively. The sequences of linker primer 1 and 8 different i5-terminal primers are shown in Table 4.

[0091] Table 4 Sequence list of special linker primers

[0092] Primer name Primer sequence 5_index sequence SEQ ID NO Adapter Primer 1 GATCGGAAGAGCCACATACTGA - SEQ ID NO: 1 Primer 1 at i5 end AATGATACGGCGACCACCGAGATCTACACCCTCTCTATACACTCTTTCCCTACACGACGCTCTTCCGATCT CTCT CTAT SEQ ID NO: 2 Primer 2 at i5 end AATGATACGGCGACCACCGAGATCTACACTATCCCTCTACACTCTTTCCCTACACGACGCTCTTCCGATCT TATC CTCT SEQ ID NO: 3 Primer 3 at the i5 end AATGATACGGCGACCACCGAGATCT...

Embodiment 2

[0106] Example 2 Kit and detection method for combined detection of sarcoma-related fusion genes or mutations

[0107] A kit for combined detection of fusion genes or mutations related to sarcoma, comprising: (1) special adapters for DNA library construction; (2) specific compound primers for detection of fusion genes and point mutations; (3) library expansion Add compound primers.

[0108] The kit above also includes: a reverse transcription reaction reagent set; a DNA fragmentation / end repair / dA addition reagent set; a DNA ligation reagent set; a PCR amplification reagent set; a high-fidelity hot-start PCR amplification reagent set.

[0109] Wherein, the reverse transcription reaction reagent set includes random primers, first-strand synthesis buffer, first-strand synthesis enzyme mixture, second-strand synthesis buffer, second-strand synthesis enzyme mixture and ultrapure water. DNA Fragmentation / End Repair / dA Addition Reagent Set includes 10×DNA Fragmentation Buffer, 5×Fr...

experiment example 1

[0216] The KIT sample was tested using the steps described in Example 2 of the present invention. The following experiments were performed with the informed consent of the patients themselves.

[0217] 1. Sample 1: The source is a gastrointestinal stromal tumor sample (No. 27698) from a patient in the General Surgery Department of Yunnan Cancer Hospital.

[0218] Test results such as Figure 9 As shown, the sample is positive for the KIT:NM_000222:exon11:c.T1727C:p.L576P mutation, and the mutation rate is 32.08%.

[0219] 2. Sample 2: The source is a sample of a healthy person undergoing physical examination by Suzhou Kono Medical Laboratory (No. 190021).

[0220] Test results such as Figure 10 As shown, the sample is negative for the KIT:NM_000222:exon11:c.T1727C:p.L576P mutation.

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Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a sarcoma fusion gene joint detection primer group, kit and detection method. The kit provided by the invention comprises: 1) a special linker for DNA library construction; 2) a specific composite primer for detecting fusion genes and point mutation; and 3) a composite primer for library amplification. DNA and RNA of a to-be-detected sample are respectively extracted, reverse transcription is performed on RNA to synthesize cDNA, the cDNA is mixed with DNA, fragmentation, terminal repair and linker ligation are performed on the mixture, PCR amplification is performed on the product, a library is enriched, and high-throughput sequencing is performed. According to the fusion gene detection kit and detection method provided by the invention, various fusion genes or mutations related to sarcoma can be detected at one time, the detection efficiency is improved, the operation is convenient, the consumption time is short, and the sensitivity is relatively high and can reach 0.1%.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a combination detection primer set and kit for sarcoma fusion genes and / or mutations and a detection method thereof. Background technique [0002] Sarcomas are malignant tumors derived from mesenchymal tissue (including connective tissue and muscle), and mostly occur in the skin, subcutaneous tissue, periosteum, and both ends of long bones. According to statistics, there are about 11,000 new sarcoma patients in the United States every year, and about 3,800 of them die from this disease, while there are about 200,000 new cases worldwide every year. Soft tissue sarcoma can occur at any age, and the most common age is 30-50 years old. There are slightly more men than women. About 60% of them occur in the limbs, 19% in the trunk, and other parts include retroperitoneum and neck. At present, there are more than 100 different subtypes showing their histological diversity. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 孙石磊姬云
Owner 苏州科贝生物技术有限公司
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