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CN-116200461-B - Library construction method for simultaneously detecting multiple genetic variation types of brain tumor, composition used by library construction method and application of library construction method

CN116200461BCN 116200461 BCN116200461 BCN 116200461BCN-116200461-B

Abstract

The invention discloses a library construction method for simultaneously detecting multiple genetic variation types of brain tumors, and a composition and application thereof. The sequencing library construction method established by the invention is a one-step library construction method, DNA mutation and RNA fusion are placed in a PCR reaction system, and pyrodetection MGMT and FISH detection CNV are optimized into an amplicon one-step method, so that mutation at the DNA level and fusion at the RNA level can be detected simultaneously, and after library construction, QPCR quantification is not needed, and only quick-on-machine sequencing operation is simple after Qubit quantification is needed. Can be used for rapidly, simply and accurately detecting the basic item of the brain tumor.

Inventors

  • SHI XIAO
  • CHEN MIN
  • LIU BAOSHI
  • ZHANG XINGXING
  • LI ZHEN
  • WANG SIZHEN
  • YAN HAI

Assignees

  • 北京泛生子基因科技有限公司

Dates

Publication Date
20260508
Application Date
20230315

Claims (6)

  1. 1. A library construction method for sequencing libraries is characterized by comprising the following steps of extracting TNA of a sample to be tested, carrying out reverse transcription by taking RNA in the TNA as a template to obtain cDNA, carrying out sulfite conversion on the TNA to obtain converted DNA, preparing a P1 primer solution containing primers, a P2 primer solution, a P3 primer solution and a P4 primer solution, carrying out PCR amplification named P1 by using the P1 primer solution to obtain a PCR product sub-library named P1 sub-library, carrying out PCR amplification named P2 by using the P2 primer solution to obtain a PCR product sub-library named P2 sub-library, carrying out PCR amplification named P3 by using the P3 primer solution to obtain a PCR product sub-library named P3 sub-library, carrying out PCR amplification named P4 by using the P4 primer solution to obtain a PCR product sub-library named P4 sub-library, mixing the P1 sub-library, the P2 sub-library, the P3 sub-library and the P4 sub-library, and carrying out PCR amplification named P4 sub-library, and carrying out mixed purification on the library to obtain a mixed library; the PCR amplified template of the P1 amplification is the cDNA, the PCR amplified template of the P3 amplification is the sulfite converted DNA, and the PCR amplified templates of the P2 amplification and the P4 amplification are the TNA; The primer of the P1 primer solution is a composition of G13f and G13r, the G13f is a mixture of 11 DNA molecules with nucleotide sequences of sequences 1-11 in a sequence table, and the G13r is a mixture of DNA molecules shown in sequences 15-25 in the sequence table; The P2 primer solution is a composition of G13f12 and G13r12, wherein G13f12 is a DNA molecule shown as a sequence 12 in a sequence table, and G13r12 is a DNA molecule shown as a sequence 26 in the sequence table; The P3 primer solution is a composition of G13f13 and G13r13, wherein G13f13 is a DNA molecule shown as a sequence 13 in a sequence table, and G13r13 is a DNA molecule shown as a sequence 27 in the sequence table; The P4 primer solution is a composition of G13f14 and G13r14, wherein G13f14 is a DNA molecule shown as a sequence 14 in a sequence table, and G13r14 is a DNA molecule shown as a sequence 28 in the sequence table; The PCR system also comprises a specific joint, wherein the specific joint is 7 DNA molecules of which the nucleotide sequences are respectively sequences 29-35 in a sequence table; The preparation method of the mixed library comprises the steps of adding 4 times of water into the P4 sub-library to dilute to obtain P4 sub-library diluent, and mixing the P1 sub-library, the P2 sub-library, the P3 sub-library and the P4 sub-library diluent to obtain a mixed library, wherein the volume ratio of the P1 sub-library, the P2 sub-library, the P3 sub-library and the P4 sub-library diluent is 5:20:20:1.5.
  2. 2. The method of claim 1, wherein the sequencing library is used to detect a type of gene mutation in brain glioma, the type of gene mutation comprising a DNA hot spot mutation, an RNA fusion mutation, an MGMT methylation mutation, a 1p/19q associative deletion and/or a chromosomal copy number variation.
  3. 3. The method according to claim 1 or 2, wherein the PCR is performed by denaturing at 95℃for 2min, stopping the reaction at 95℃for 30s,60℃for 90s,72℃for 90s,23 cycles, and 72℃for 10 min.
  4. 4. The method according to claim 1 or 2, wherein the sample to be measured is a paraffin sample.
  5. 5. The composition for detecting the primer and the linker of the glioma gene is characterized by comprising DNA molecules shown as sequence 1-sequence 35 in a sequence table.
  6. 6. Use of the composition of claim 5 for the preparation of a gene product for detecting glioma.

Description

Library construction method for simultaneously detecting multiple genetic variation types of brain tumor, composition used by library construction method and application of library construction method Technical Field The invention relates to the field of biotechnology, in particular to a library construction method for simultaneously detecting multiple genetic variation types of brain tumors, and a composition and application thereof. Background Gliomas are the most common primary intracranial malignancy, with a high degree of heterogeneity, poor prognosis. With the development of brain tumor genomics in recent years, molecular pathology detection using genes as markers has become a well-accepted trend in the medical community as a premise and basis for glioma treatment. Numerous clinical studies have also demonstrated that specific genes are directly related to tumor typing and drug efficacy. The WHO has incorporated molecular pathology into the glioma pathology diagnostic system in 2016. Molecular pathology based on tumor genetic level can more accurately judge the clinical prognosis of glioma patients, and provides a differentiation basis for tumors which are difficult to clearly diagnose and classify by histology. The basic items of brain tumor comprise DNA hot spot mutation (such as IDH mutation, TERT promoter mutation, BRAF mutation, ATRX mutation and the like), RNA fusion, MGMT methylation, 1p/19q combined deletion, and Chr7 and Chr10 copy number mutation, and the DNA hot spot mutation can be performed by a first generation method and a second generation method of NGS at present. RNA fusion is generally carried out by the method of FISH and NGS, MGMT methylation detection is carried out by the method of pyrosequencing, and 1p/19q, chr7 and Chr10 copy number variations are detected by the method of FISH. In 1 sample, all the items are detected, the required initial sample quantity is large, the method is complicated, and the requirements on the sample and technicians and laboratory conditions are relatively high. Therefore, there is a need to design a new glioma detection method to solve the above-mentioned problems. TNA includes DNA and RNA, and is simply called as the sum of the two. Disclosure of Invention The technical problem to be solved by the invention is how to rapidly, simply and/or accurately detect marker genes of glioma or detect basic items of glioma. In order to solve the technical problems, the invention firstly provides a method for sequencing library construction. The method comprises the steps of extracting TNA of a sample to be detected, carrying out reverse transcription on the TNA to obtain cDNA, carrying out sulfite conversion on the TNA to obtain converted DNA, preparing a P1 primer solution containing primers, a P2 primer solution, a P3 primer solution and a P4 primer solution, carrying out PCR amplification named P1 sub-library by using the P1 primer solution to obtain a PCR product sub-library named P1 sub-library, carrying out PCR amplification named P2 sub-library by using the P2 primer solution to obtain a PCR product sub-library named P2 sub-library, carrying out PCR amplification named P3 sub-library by using the P3 primer solution to obtain a PCR product sub-library named P3 sub-library, carrying out PCR amplification named P4 sub-library by using the P4 primer solution to obtain a PCR product sub-library named P4 sub-library, mixing the P1 sub-library, the P2 sub-library, the P3 sub-library and the P4 sub-library, and carrying out PCR amplification named P4 sub-library, and carrying out sequencing library mixing to obtain a sequencing library. In the method, 1 part of TNA is used for carrying out reverse transcription to obtain the cDNA for carrying out the P1 amplification, 1 part of TNA is used for carrying out sulfite conversion to obtain the converted DNA for carrying out the P3 amplification, and the rest 2 parts of TNA can be directly used for carrying out the P2 amplification and the P4 amplification without treatment. In the above method, the PCR amplified template for the P1 amplification may be the cDNA. The P3 amplified PCR amplified template may be the sulfite converted DNA. The P2 amplified and the P4 amplified PCR amplified templates may be the TNA. In the methods described above, the sequencing library may be used to detect the type of gene mutation in brain glioma. The types of genetic mutations may include DNA hot spot mutations, RNA fusion mutations, MGMT methylation mutations, 1p/19q associative deletions, and/or chromosomal copy number variations. The DNA hotspot mutated genes may include IDH1, IDH2, BRAF, H3F3A, HIST1H3B, HIST H3C, TERT genes. The RNA fusion mutated gene may include KIAA1549-BRAF. The methylation mutant gene can include MGMT. The copy number variation may include 1p/19q and/or chr7 and chr10 copy number variation. In the above method, the primer of the P1 primer solution is a combination of G13f and G13 r. The G13f can be a mixtur