CN111378752A - POLN gene mutation site detection kit and application thereof - Google Patents
POLN gene mutation site detection kit and application thereof Download PDFInfo
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- CN111378752A CN111378752A CN202010212334.0A CN202010212334A CN111378752A CN 111378752 A CN111378752 A CN 111378752A CN 202010212334 A CN202010212334 A CN 202010212334A CN 111378752 A CN111378752 A CN 111378752A
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Abstract
The invention belongs to the technical field of biological detection, and relates to a detection kit for a POLN gene mutation site and application thereof. The detection kit comprises an amplification primer and an extension primer, wherein the sequence of a forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of a reverse primer of the amplification primer is shown as SEQ ID NO.4, the sequence of the extension primer is shown as SEQ ID NO.5, the sequence of the POLN gene is shown as SEQ ID NO.1, and the sequence of the mutated POLN gene is shown as SEQ ID NO. 2. The POLN gene mutation site detection kit can predict the radiation sensitivity of personnel with high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a detection kit for a POLN gene mutation site and application thereof.
Background
Currently, radiation therapy remains the mainstay of modern cancer therapy, and approximately 50% of cancer patients require radiation therapy. However, patients receiving the same dose of radiation therapy exhibit different radiotoxicity: few have no obvious toxic effects, most have mild or moderate toxic reactions clinically, while few cause severe normal tissue complications and may even be life threatening. Therefore, there is a need to find a molecular marker associated with radiosensitivity for predicting whether a patient is a radiosensitive (or radioresistant) individual.
The prediction of radiation sensitivity is to be able to tailor the radiation treatment regimen to individual patients to improve prognosis, so that on the one hand the radiation dose can be reduced to reduce toxicity to sensitive individuals, and on the other hand the radiation dose can be increased to allow more radiation resistant patients to be effectively treated. This will maximize control of the tumor while minimizing damage to the patient's normal tissues. In addition, about 20 thousands of radiation workers exist in China, the radiation workers need to receive professional irradiation for a long time during daily work, and prediction of radiation sensitivity of the radiation workers is helpful for preventing and reducing professional injuries.
The locus POLN rs2022302 is located on chromosome 4, 2174006, the allele is a > G, and the gene in which the locus is located encodes a member of the type a family of DNA polymerases. The encoded proteins play a role in DNA repair and homologous recombination.
Single nucleotide polymorphism markers (SNPs) are "third generation DNA genetic markers", and 300 ten thousand of them exist in the human genome, and are considered as genetic markers with the best application prospect. SNPs can truly reflect genetic differences and are associated with radiation-induced toxicity in normal tissues.
In addition, compared with the traditional gene detection method mainly based on sequencing and hybridization principles, the matrix-assisted laser desorption ionization mass spectrometry technology has incomparable advantages in the aspects of detection efficiency, detection flux, detection sensitivity, detection accuracy, detection repeatability, detection cost and the like. The technology can realize simultaneous detection of 384 samples, the detection of each detection point only needs 3-5s, pmol level can be detected, and the accuracy reaches more than 98%. Therefore, the matrix-assisted laser desorption ionization mass spectrometry is used for judging the radiation sensitivity, so that the personnel radiation sensitivity can be quickly, accurately, high in flux and low in cost.
Disclosure of Invention
The invention aims to provide a detection kit for a POLN gene mutation site, which can predict the radiation sensitivity of personnel with high efficiency and low cost.
To achieve the object, in a basic embodiment, the present invention provides a detection kit for a mutant site of a POLN gene, the detection kit comprising an amplification primer and an extension primer,
the sequence of the forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of the reverse primer of the amplification primer is shown as SEQ ID NO.4,
the sequence of the extension primer is shown as SEQ ID NO.5,
the sequence of the POLN gene is shown as SEQ ID NO.1, the mutation site is rs2022302 site (mutation from A to G) of the POLN gene, and the sequence of the mutated POLN gene is shown as SEQ ID NO. 2.
In a preferred embodiment, the present invention provides a kit for detecting a mutant site of a POLN gene, wherein the concentration of the forward primer of the amplification primer is 2-3. mu.M.
In a preferred embodiment, the present invention provides a kit for detecting a mutant site of a POLN gene, wherein the concentration of the reverse primer of the amplification primer is 2 to 3 μ M.
In a preferred embodiment, the present invention provides a kit for detecting a mutant site of a POLN gene, wherein the concentration of the extended primer is 3.5 to 4.5. mu.M.
The second purpose of the invention is to provide the application of the detection kit for preparing the kit for predicting the radiation sensitivity of the personnel, so that the radiation sensitivity of the personnel can be predicted with high efficiency and low cost.
To achieve this object, in a basic embodiment, the present invention provides the use of the above-described detection kit for the preparation of a kit for predicting the radiation sensitivity of a person.
The detection kit for the POLN gene mutation site has the advantages of high efficiency and low cost, and can predict the radiation sensitivity of personnel.
Detailed Description
The following examples further illustrate the practice of the present invention, but the embodiments of the present invention are not limited to the following examples.
Example 1:
1) sample acquisition and irradiation and chromosome aberration analysis
Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy60And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a susceptible group, a general group and an insensitive group. After the irradiation, the blood sample is cultured for 52h, and then the chromosome is harvested and sliced. Each sample was analyzed for 200 metaphase phases.
2) Peripheral blood genome DNA extraction
Genomic DNA of 0Gy irradiated samples of susceptible and non-susceptible groups was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.
3) Whole exon capture sequencing
Sequencing data is firstly subjected to data filtration to remove low-quality data, and clear Reads are obtained. The sequencing needs to reach the clean Reads rate of more than 90%, the clean base rate of more than 20G, the clean base rate of more than 90%, and the Q20 rate of more than 98%, and the experimental sample meets the requirements.
4) Biological information analysis
Clear Reads were aligned to the reference genome and differential SNP sites were screened. Reference genome version: GRCh37(hg19), ftp:// ftp.1000genes.ebi.ac.uk/vol 1/ftp/technical/reference/human _ g1k _ v37. fasta.gz. The locus of the differential locus, POLNrs2022302, was selected by bioinformatic analysis, as shown in table 1.
TABLE 1 selected radiation sensitive sites
SNP site | Gene | SNP site position | Alleles |
rs2022302 | POLN | chr4:2174006 | A/G |
Example 2:
1) the method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) human whole blood genome (blood from healthy male volunteers between 20-30 years old) DNA extraction was performed according to the product instructions.
2) The amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 is adopted, and a specific reaction system (5 mu l of the reaction system comprises 0.95 mu l H)2O、0.625μl PCR Buffer(10×)、0.325μl MgCl2PCR reaction (25mM), 1. mu.l dNTP (2.5mM), 1. mu.l primer, 0.1. mu.l HotstarTaq (5U/. mu.L)) was performed according to the following reaction program: 15min at 94 ℃; [94 ℃, 20sec, 56 ℃, 30sec ]]45 cycles; 72 ℃ for 4 min. The reaction product was stored at 4 ℃.
3) Using an SAP reaction solution (2. mu.l SAP reaction solution included 1.53. mu.l H)2O, 0.17. mu.l of SAP Buffer (10 ×), 0.3. mu.l of SAP enzyme (1U/. mu.L)) were applied to the reaction product of step 2) at 37 ℃ for 40min and 85 ℃ for 5min, and the treated product was stored at 4 ℃.
4) Carrying out extension reaction on the treated product in the step 3) by adopting an extension primer of SEQ ID NO.5,
2 μ l reaction included 0.755 μ l H2O, 0.2. mu.l of iPLex Buffer (10 ×), 0.2. mu.l of iPLEX termination mix, 0.041. mu.l of iPLex enzyme, 0.804. mu.l of primer.
The reaction procedure is as follows: 30s at 94 ℃; 5 cycles of [94 ℃, 5s, (52 ℃, 5s, 80 ℃, 5s) ]40 cycles; 72 ℃ for 3 min. The extension product was stored at 4 ℃.
5) Purifying the extension product of step 4). 6mg of resin was uniformly applied to cover the 384 well plate and left for 20 min. The 384 well plate containing the extension product of step 4) was centrifuged at 1000rpm for 1min, 25. mu.L of deionized water was added to each well, inverted on the resin plate, and then the resin plate was snapped on the 384 well plate in the inverted position, and the resin was dropped into the 384 well plate by tapping, and the membrane was sealed. The 384 well plate was inverted for 20 minutes with the long axis of the 384 well plate as the axis, and centrifuged at 3500rpm for 5 minutes for use.
6) Detecting the genotype of the gene locus: the sample treated in step 5) was transferred to a MassARRAYPectroCHIP chip (SAMSUNG, MassArray. TM. Nanodispenser) and put into a mass spectrometer (SEQUENOM, MassARRAY compact System) for detection.
The detection result shows that the primer designed in the embodiment can detect the genotype of the locus rs2022302 of the POLN gene and can be used for detecting the mutation of the locus rs2022302 of the radiation-sensitive gene.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.
Sequence listing
<110> China institute for radiation protection
<120> detection kit for POLN gene mutation site and application thereof
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<141>2020-03-24
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gggacaatgt ccctgtaaga aaaacgtaac ttttgaggag gatcctgagg agccattaaa 240
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Claims (5)
1. A detection kit for a POLN gene mutation site is characterized in that: the detection kit comprises an amplification primer and an extension primer,
the sequence of the forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of the reverse primer of the amplification primer is shown as SEQ ID NO.4,
the sequence of the extension primer is shown as SEQ ID NO.5,
the sequence of the POLN gene is shown as SEQ ID NO.1, and the sequence of the mutated POLN gene is shown as SEQ ID NO. 2.
2. The detection kit according to claim 1, characterized in that: the concentration of the forward primer of the amplification primer is 2-3 mu M.
3. The detection kit according to claim 1, characterized in that: the concentration of the reverse primer of the amplification primer is 2-3 mu M.
4. The detection kit according to claim 1, characterized in that: the concentration of the extension primer is 3.5-4.5 mu M.
5. Use of the test kit according to any one of claims 1 to 4 for the preparation of a kit for predicting the radiation sensitivity of a human.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111286540A (en) * | 2020-03-24 | 2020-06-16 | 中国辐射防护研究院 | Use of POLN gene as molecular marker for predicting radiation sensitivity |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110878357A (en) * | 2019-12-06 | 2020-03-13 | 苏州卫生职业技术学院 | Probe library, detection method and kit for detecting effectiveness of DNA cross-damage synthesis repair pathway |
CN111286540A (en) * | 2020-03-24 | 2020-06-16 | 中国辐射防护研究院 | Use of POLN gene as molecular marker for predicting radiation sensitivity |
EP3778924A1 (en) * | 2019-08-16 | 2021-02-17 | Siemens Healthcare GmbH | Molecular predictors of patient response to radiotherapy treatment |
-
2020
- 2020-03-24 CN CN202010212334.0A patent/CN111378752A/en active Pending
Patent Citations (3)
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EP3778924A1 (en) * | 2019-08-16 | 2021-02-17 | Siemens Healthcare GmbH | Molecular predictors of patient response to radiotherapy treatment |
CN110878357A (en) * | 2019-12-06 | 2020-03-13 | 苏州卫生职业技术学院 | Probe library, detection method and kit for detecting effectiveness of DNA cross-damage synthesis repair pathway |
CN111286540A (en) * | 2020-03-24 | 2020-06-16 | 中国辐射防护研究院 | Use of POLN gene as molecular marker for predicting radiation sensitivity |
Non-Patent Citations (4)
Title |
---|
1000GENOMES: "ss111661519" * |
GEORGE-LUCIAN MOLDOVAN: "DNA Polymerase POLN Participates in Cross-Link Repair and Homologous Recombination" * |
NCBI: "Homo sapiens DNA polymerase nu (POLN), RefSeqGene on chromosome 4" * |
陈荣: "射线 DNA损伤修复酶基因多态性与福建地区 急性白血病的相关性:病例对照研究" * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111286540A (en) * | 2020-03-24 | 2020-06-16 | 中国辐射防护研究院 | Use of POLN gene as molecular marker for predicting radiation sensitivity |
CN111286540B (en) * | 2020-03-24 | 2023-05-12 | 中国辐射防护研究院 | Use of POLN gene as molecular marker for predicting radiation sensitivity |
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