Method for analyzing early screening of ovarian cancer based on XRCC3 and XPC genes

Abstract

The invention discloses an early screening method for ovarian cancer analysis based on XRCC3 and XPC genes, a new biomarker is provided for early diagnosis of ovarian cancer by identifying gene polymorphism, the method comprises the following steps: collecting peripheral blood samples from clinical ovarian cancer patients and a control group, extracting whole genome DNA and purifying, then performing gene hybridization by using a 22K Human Genome Array chip, and finally identifying the ovarian cancer. Specific polymorphic sites of the XRCC3 and XPC genes are analyzed, after amplification, fragmentation and hybridization, a high-resolution fluorescence image is obtained by using a laser scanner, gene expression differences are evaluated through image analysis software, finally, significantly differential genes are verified through an RT-PCR technology, and early diagnosis of ovarian cancer is facilitated through the specific polymorphic sites of the XRCC3 and XPC genes.

Description

Technical Field

[0001] The present invention relates to the technical field of XXXXX, and in particular to an early screening method for ovarian cancer based on XRCC3 and XPC gene analysis. Background Art

[0002] Ovarian cancer is one of the most lethal malignant tumors in the female reproductive system. Early diagnosis is crucial to improving patient survival. Studies have shown that abnormalities in the DNA repair mechanism are closely related to the development of various cancers, especially ovarian cancer. XRCC3 and XPC genes play a key role in DNA repair. XRCC3 is involved in homologous recombination repair, while XPC plays an important role in nucleotide excision repair. Genetic polymorphisms of the two may affect an individual's ability to repair DNA damage, thereby increasing the risk of ovarian cancer.

[0003] In recent years, studies have shown that DNA repair mechanisms play an important role in the occurrence and development of ovarian cancer; DNA repair genes such as XRCC3 and XPC have key functions in repairing DNA damage, involving the cell's response to environmental factors and endogenous damage; the protein encoded by the XRCC3 gene is mainly involved in homologous recombination repair, responsible for repairing double-stranded DNA breaks; while the protein encoded by the XPC gene plays a role in nucleotide excision repair, responsible for identifying and repairing DNA damage caused by ultraviolet rays and chemicals;

[0004] The polymorphisms of these genes may lead to changes in repair capacity, thus affecting an individual's susceptibility to cancer. For example, certain single nucleotide polymorphisms (SNPs) in the XRCC3 gene have been shown to be associated with the risk of ovarian cancer. Similarly, polymorphisms in the XPC gene are also closely associated with the occurrence of a variety of cancers. Although studies have explored the relationship between polymorphisms in the XRCC3 and XPC genes and ovarian cancer, there is a lack of systematic methods for effectively detecting these polymorphisms as biomarkers for early screening.

[0005] Existing ovarian cancer screening methods mostly rely on imaging examinations and CA-125 marker testing, but these methods have limited sensitivity and specificity in early detection;

[0006] Based on this, early screening methods for ovarian cancer based on XRCC3 and XPC gene analysis are needed. Summary of the invention

[0007] To achieve the above object, the present invention provides the following scheme: an early screening method for ovarian cancer based on XRCC3 and XPC gene analysis, comprising the following steps:

[0008] Step 1: 40 peripheral blood samples were collected from the experimental group (clinical ovarian cancer patients) and the control group (patients with other benign diseases) to provide basic samples for XRCC3 and XPC gene detection;

[0009] Step 2: Use a DNA extraction kit to extract whole genome DNA from the collected blood samples, purify it using a DNAClean&Concentrator TM-5 kit to remove inhibitory factors and contaminants, and then measure the DNA concentration using a NanoDrop UV spectrophotometer. Use 0.7% agarose gel for electrophoresis to screen qualified DNA samples;

[0010] Step 3, preparing a 22K Human Genome Array chip, the chip comprising 21522 oligo DNA probes, wherein the specific probes are used to identify the A4541G, A17893G, T241M sites of the XRCC3 gene and the Ala499Val, Lys939Gln sites of the XPC gene, each probe specifically identifies a human gene transcript, and reversely transcribes the mRNA of the experimental group - clinical ovarian cancer patients and the normal control group - other benign diseases into cDNA, and uses the cDNA as a probe to hybridize with the XRCC3 and XPC specific probes on the chip;

[0011] Step 4: Quantitative analysis of the extracted DNA samples was performed and standardized to 50 ng / μL. The amount of DNA sample required for chip analysis was 200 ng. 0.1 N NaOH was added to the sample to denature the DNA into a single strand, which then combined with the XRCC3 and XPC gene-specific probes on the chip. Subsequently, the neutralized sample was added to the whole genome amplification reagent and incubated overnight at 37°C to make the total amount of amplified DNA reach 2000-3000 times the initial loading amount.

[0012] Step 5: The amplified DNA sample is subjected to controlled enzymatic treatment so that the fragmentation process does not damage the integrity of the XRCC3 and XPC genes. After fragmentation, the fragmented DNA is precipitated by adding isopropanol and centrifuged at 4°C to enrich the fragmented DNA, and then dried in air and resuspended in hybridization buffer;

[0013] Step 6: Hybridize the DNA sample resuspended in hybridization buffer with the prepared chip and place it in a hybridization oven for overnight reaction. At this time, the fragmented DNA effectively combines with the XRCC3 and XPC gene site-specific probes on the chip. The chip contains a total of 610,000 types of microbeads, each of which corresponds to the detection of one SNP site.

[0014] Step 7: Wash and remove the unhybridized and non-specifically hybridized DNA, retain only the DNA bound to the XRCC3 and XPC gene sites, use the captured DNA to perform a single base extension reaction on the chip, and add a detectable label group on the chip to distinguish the SNP types of different samples in the XRCC3 and XPC genes, and place the chip after the reaction in the XC4 reagent for coating to form a protective layer to stabilize the detection signals of the XRCC3 and XPC genes;

[0015] Step 8: Scan the chip using a LuxScan 10KA dual-channel laser scanner to excite the fluorescent groups of the single-base extension products on the chip to generate high-resolution images. The obtained data are imported into LuxScan 3.0 image analysis software to analyze the fluorescence signal intensity and ratio between OC tissue and normal tissue, and 12 housekeeping genes are used for balance.

[0016] In the data analysis, the criteria for determining the differential expression of XRCC3 and XPC genes were set as follows:

[0017] Standard 1: The ratio of OC tissue to normal tissue must be greater than 2.0 or less than 0.5, and the gene expression change is more than 2 times, reflecting a significant difference;

[0018] Standard 2: The signal intensity of either OC tissue or normal tissue must be greater than 800, or both must be greater than 200, and the dominant expression data can be repeated in all samples;

[0019] Step 9. Select one up-regulated gene and one down-regulated gene with significant difference in ratio value in XRCC3 and XPC genes in the gene expression spectrum, and verify them by RT-PCR technology to confirm the difference in expression of XRCC3 and XPC genes in ovarian cancer patients.

[0020] Further preferably, in the step three, the reverse transcription reaction of the mRNA adopts CbcScript reverse transcriptase to ensure the reverse transcription efficiency and the integrity of cDNA, by mixing the mRNA collected from the experimental group - clinical ovarian cancer patients and the control group - patients with other benign diseases, and setting the reaction conditions provided in the user manual of CbcScript reverse transcriptase, including appropriate temperature and time. After the reaction is completed, the quality of cDNA is confirmed by agarose gel electrophoresis, and the integrity is verified by comparing the band size of cDNA with the standard marker.

[0021] Further preferably, in step 4, the concentration range of the whole genome amplification reagent added is 1-5 μL, and the volume of the reaction system is maintained at 20-50 μL, and a thermal cycle program is used during the amplification process, including the following stages:

[0022] Denaturation stage: keep at 95℃ for 30 seconds to 1 minute to completely separate the double-stranded DNA; Annealing stage: lower the temperature to 55℃-65℃ and keep for 30 seconds to 1 minute to allow the primer to specifically bind to the target sequence; Extension stage: set the temperature at 72℃ and keep for 30 seconds to 1 minute to amplify the target sequence using Taq DNA polymerase;

[0023] After amplification, the concentration of the amplified product was measured by a photometer, and the amount of DNA sample obtained met the 200 ng standard required for subsequent chip analysis.

[0024] Further preferably, in step five, the fragmented DNA sample needs to be subjected to enzymatic treatment, and the enzyme selected includes restriction endonuclease or DNA fragmentase, and the generated DNA fragments are in the range of 200-500 bp. After DNA precipitation, unbound reagents and enzymes are removed by centrifugation and washing to obtain a high-purity fragmented DNA sample, which is then resuspended in an appropriate hybridization buffer.

[0025] Further preferably, in step six, the fragmented DNA sample is denatured to convert it into a single-stranded form. At this time, the sequence in the DNA sample is annealed with the site-specific 50-base probes on the chip. Each microbead is connected to a different 50-base sequence. The chip contains a total of 610,000 types of microbeads, and each microbead corresponds to the detection of one SNP site.

[0026] Further preferably, in step seven, the chip is placed in an XC4 reagent for coating, so that the surface of the chip is coated with a layer of viscous transparent liquid to form a protective layer outside the chip.

[0027] Further preferably, in step eight, the chip treated with hybridization and extension reaction is placed in a scanner, and the fluorescent group of the single-base extension product on the chip is excited by laser. The scanner obtains the fluorescent signal emitted by the fluorescent group and generates high-resolution image data. The obtained image data is directly imported into LuxScan3.0 image analysis software for processing and analysis, and the SNP typing data of each sample is extracted.

[0028] Further preferably, in the step nine, specific primers are designed according to the sequence information of the target gene, and the Tm values ​​of the primers are similar. In the RT-PCR reaction, the initial denaturation temperature is set to 95°C, the annealing temperature is 60°C, the extension temperature is 72°C, and each cycle is set to 30 seconds. The PCR products are separated by agarose gel electrophoresis to confirm the specificity and size of the amplification, and the expression differences of the up-regulated and down-regulated genes are confirmed by comparing the band intensity and position of the amplified products.

[0029] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0030] The present invention uses SNP gene chip technology to systematically study the relationship between the polymorphism of XRCC3 and XPC genes and the incidence of ovarian cancer. Through high-throughput gene detection, specific polymorphic sites related to ovarian cancer can be efficiently identified, thereby providing more accurate biomarkers for early screening of ovarian cancer. In addition, the implementation method of the present invention ensures high-quality acquisition of samples and accuracy of data analysis, has the advantages of simple operation, good repeatability and high sensitivity, and contributes to the early diagnosis of ovarian cancer through specific polymorphic sites of XRCC3 and XPC genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is a flow chart of the steps of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Example

[0036] like Figure 1 As shown, the early screening method for ovarian cancer based on XRCC3 and XPC gene analysis in this embodiment includes the following steps:

[0037] 40 peripheral blood samples were collected from the experimental group - patients with clinical ovarian cancer. The patients had to be diagnosed by doctors. At the same time, 40 peripheral blood samples were collected from the control group - patients with other benign diseases to ensure the comparability between samples. During the sample collection process, an appropriate amount of peripheral blood (5-10 ml) was drawn from the vein of each participant using aseptic techniques. The samples were then immediately placed in EDTA anticoagulant tubes to prevent blood coagulation. The collected samples were stored at 4°C and sent to the laboratory for subsequent processing.

[0038] Whole genome DNA was extracted from the collected blood samples using a DNA extraction kit, purified using a DNA Clean&Concentrator TM-5 kit to remove inhibitory factors and contaminants, and then the DNA concentration was measured using a NanoDrop UV spectrophotometer. 0.7% agarose gel electrophoresis was used to screen qualified DNA samples.

[0039] Specifically, the collected peripheral blood sample is transferred to a centrifuge tube, and centrifuged according to the instructions of the DNA extraction kit to separate plasma and cell components. Subsequently, the plasma portion is removed, the cell pellet is retained, and an appropriate amount of lysis buffer is added to mix thoroughly to destroy the cell membrane and release the intracellular DNA. Proteinase K is added to digest the intracellular protein and purify the DNA. After the cell lysis is completed, the DNA Clean&ConcentratorTM-5 kit is used for purification. According to the instructions of this kit, the lysate is transferred to the column, and the DNA is bound to the membrane of the column by centrifugation. Subsequently, the unbound impurities and inhibitory factors are washed away by the washing buffer to ensure the high purity of the extracted DNA. Finally, the purified DNA is eluted with the elution buffer and collected in a new centrifuge tube. The concentration of the extracted whole genome DNA is then determined by the NanoDrop ultraviolet spectrophotometer. An appropriate amount of DNA sample is dropped on the measuring table, and the concentration and purity of the DNA are automatically calculated by measurement, and its purity is evaluated by the A260 / A280 ratio. The ideal value should be between 1.8 and 2.0 to ensure the quality of the extracted DNA.

[0040] In order to further confirm the integrity of the DNA sample, 0.7% agarose gel was used for electrophoresis detection. The agarose was mixed with electrophoresis buffer and heated to dissolve it. After cooling, it was poured into the electrophoresis tank to form a gel. DNA loading buffer was added to the gel, and the extracted DNA sample was added to the gel well. Electrophoresis was performed under the action of an electric field. The DNA fragments were separated in the gel by electrophoresis and stained with the fluorescent dye SYBR Green. After the electrophoresis, the gel image was observed and photographed using an ultraviolet light to analyze the size and integrity of the DNA bands.

[0041] A 22K Human Genome Array chip was prepared, the chip comprising 21,522 oligo DNA probes, each probe specifically identifying a human gene transcript, and the mRNA of the experimental group - clinical ovarian cancer patients and the mRNA of the normal control group - other benign diseases were reverse transcribed into cDNA, which was hybridized with the chip as a probe; the reverse transcription reaction of the mRNA used CbcScript reverse transcriptase to ensure the reverse transcription efficiency and the integrity of the cDNA, by mixing the mRNA collected from the experimental group and the control group, and setting the reaction conditions provided in the user manual of the CbcScript reverse transcriptase, including appropriate temperature and time, and after the reaction was completed, the quality of the cDNA was confirmed by agarose gel electrophoresis, and the integrity was verified by comparing the band size of the cDNA with that of the standard marker.

[0042] The extracted DNA samples were quantitatively analyzed and standardized to 50 ng / μL. The amount of DNA sample required for chip analysis was 200 ng. 0.1 N NaOH was added to the sample to denature the DNA into a single strand. After neutralization, the whole genome amplification reagent was added and incubated overnight at a constant temperature of 37°C to make the total amount of amplified DNA reach 2000-3000 times the initial loading amount. The concentration range of the whole genome amplification reagent added was 1-5 μL, and the volume of the reaction system was kept at 20-50 μL. A thermal cycle program was used during the amplification process, including a denaturation phase: maintaining at 95°C for 30 seconds to 1 minute, an annealing phase: lowering the temperature to 55°C-65°C for 30 seconds to 1 minute, and an extension phase: setting the temperature at 72°C for 30 seconds to 1 minute. The target sequence was amplified using Taq DNA polymerase. After the amplification, the concentration of the amplified product was measured by a photometer, and the amount of DNA sample obtained met the 200 ng standard required for subsequent chip analysis.

[0043] The amplified DNA sample is subjected to controlled enzymatic treatment so that the fragmentation process does not damage the integrity of the DNA. It is then precipitated by adding isopropanol and centrifuged at 4°C to enrich the fragmented DNA, which is then dried in air and resuspended in an appropriate hybridization buffer. The fragmented DNA sample needs to be enzymatically treated, and the enzyme selection includes restriction endonucleases or DNA fragmentases. The generated DNA fragments are in the range of 200-500bp. After DNA precipitation, unbound reagents and enzymes are removed by centrifugation and washing to obtain high-purity fragmented DNA samples, which are then resuspended in an appropriate hybridization buffer.

[0044] The DNA sample resuspended in the hybridization buffer was hybridized with the prepared chip and placed in a hybridization oven for overnight reaction to effectively bind the fragmented DNA to the XRCC3 and XPC gene site-specific probes on the chip; the fragmented DNA sample was denatured to convert it into a single-stranded form, at which point the sequence in the DNA sample annealed with the site-specific 50-base probe on the chip. Each bead was connected to a different 50-base sequence. The chip contained a total of 610,000 types of beads, and each bead detected a SNP site.

[0045] Specifically, the DNA sample resuspended in the hybridization buffer is hybridized with the prepared 22K Human Genome Array chip. The key to this process is to ensure that the fragmentation degree and quality of the DNA sample are suitable for effective binding with the specific probe on the chip. First, the fragmented DNA sample is denatured by heating it to 95°C for several minutes to convert the double-stranded DNA into a single-stranded form, because single-stranded DNA can better bind to the probe. The prepared chip contains 50-base-long specific probes for different target sequences. Each microbead is connected to a different 50-base sequence. The chip contains a total of 610,000 microbead types. Each microbead corresponds to the detection of a SNP site, so that each microbead can specifically capture the corresponding single-stranded DNA sequence. During the process of A sample and chip, ensure that the two are fully mixed in a suitable hybridization buffer to promote the binding between the probe and the target DNA sequence. Subsequently, place the chip in a hybridization oven and set the temperature to 55-65°C and the reaction time (overnight) to ensure sufficient time for the fragmented single-stranded DNA to effectively anneal and bind to the probe on the chip. The hybridization oven provides a constant temperature and environment, which helps to improve the binding efficiency and specificity. In this process, the reaction conditions of hybridization must be strictly controlled to avoid nonspecific binding and improve the accuracy of the signal. After overnight hybridization, the chip will be ready to enter the next step of cleaning and signal detection to ensure the performance of the chip so that it can effectively identify and analyze the polymorphism of the target gene.

[0046] The unhybridized and non-specifically hybridized DNA is removed by washing, and the captured DNA is used to perform a single base extension reaction on the chip. A detectable label group is added to the chip to distinguish the SNP types of different samples. The chip after the reaction is completed is placed in the XC4 reagent for coating, so that the surface of the chip is coated with a layer of viscous transparent liquid to form a protective layer outside the chip;

[0047] Specifically, the hybridized chip is placed in a washing buffer and washed by gentle shaking or centrifugation to ensure that all unbound DNA molecules are removed, because residual unbound DNA may interfere with subsequent signal detection and analysis. After washing, ensure that only specifically bound DNA is retained on the chip. By using the captured specific DNA to perform a single base extension reaction on the chip, in the single base extension reaction, DNA polymerase and labeled nucleotides are added to extend a single base next to each bound probe, which can ensure that the SNP type of each sample is clearly identified. By adding the label group, the subsequent signal detection is more sensitive and reliable. After the extension reaction, each SNP site on the chip will be labeled, thereby providing a basis for subsequent detection. After the reaction is completed, the chip is placed in the XC4 reagent for coating, so that the surface of the chip is coated with a layer of viscous transparent liquid. The coating layer not only helps to protect the signal on the chip and prevent its loss in subsequent operations, but also can improve the stability of the signal and the sensitivity of detection. Finally, the chip coated with the viscous transparent liquid is placed in a vacuum environment for one hour to ensure that the coating layer is evenly distributed to form an effective protective film.

[0048] The chip was scanned using a LuxScan 10KA dual-channel laser scanner. The laser excited the fluorescent groups of the single-base extension products on the chip to generate high-resolution images. The obtained data was imported into LuxScan3.0 image analysis software to analyze the fluorescence signal intensity and ratio between OC tissue and normal tissue. 12 housekeeping genes were used for balance. In the data analysis, the criteria for determining differential gene expression were set as follows:

[0049] Standard 1: The ratio of OC tissue to normal tissue must be greater than 2.0 or less than 0.5, and the gene expression change is more than 2 times, reflecting a significant difference;

[0050] Standard 2: The signal intensity of either OC tissue or normal tissue must be greater than 800, or both must be greater than 200, and the dominant expression data can be repeated in all samples;

[0051] Specifically, use LuxScan The 10KA dual-channel laser scanner scans the processed chip to obtain the fluorescence signal of each SNP site in the sample. The chip is carefully placed in the sample slot of the scanner to ensure its alignment with the laser path to avoid position deviation during the scanning process. In the scanning setting, the laser wavelength used should match the fluorescent marker to improve the intensity and clarity of the fluorescent signal. The scanning process will last for a period of time. The laser scans the chip line by line, and the fluorescent signal released by each microbead is captured by the detector and converted into an electrical signal. The scanner setting should ensure high resolution to accurately identify and record the fluorescence intensity of each SNP site. During the entire scanning process, the equipment will monitor and record data in real time and generate image files. After the scan is completed, the obtained image data is imported into LuxScan3.0 image analysis software for subsequent analysis. The image analysis software can process and analyze the scanned images and extract the fluorescence signal intensity and ratio between OC tissue and normal tissue. To ensure the accuracy of the data, 12 housekeeping genes are used as internal reference genes for balanced correction during the analysis process. The expression of these housekeeping genes is relatively stable under different samples and experimental conditions, which helps to eliminate signal fluctuations caused by differences between samples or experimental errors.

[0052] In data analysis, the criteria for determining differential gene expression were set to evaluate changes in gene expression between OC tissues and normal tissues;

[0053] The first criterion is that the ratio of OC tissue to normal tissue must be greater than 2.0 or less than 0.5, indicating that the gene expression change is more than 2 times, thus reflecting a significant difference. This criterion ensures that genes with significantly upregulated or downregulated expression in ovarian cancer patients can be screened as potential biomarkers.

[0054] Standard 2 requires that the intensity of either OC tissue or normal tissue signal must be greater than 800, or both must be greater than 200, and the dominant expression data can be repeated in all samples. The setting of this standard can ensure the reliability of the results and avoid false positive results caused by accidental fluctuations.

[0055] One up-regulated gene and one down-regulated gene with significant differences in ratio values ​​were selected from the gene expression profile, and the differences in gene expression were confirmed by RT-PCR technology. Specific primers were designed based on the sequence information of the target gene, and the Tm values ​​of the primers were similar. In the RT-PCR reaction, the initial denaturation temperature was set to 95°C, the annealing temperature was set to 60°C, the extension temperature was set to 72°C, and each cycle was set to 30 seconds. The PCR products were separated by agarose gel electrophoresis to confirm the specificity and size of the amplification. The expression differences of the up-regulated and down-regulated genes were confirmed by comparing the band intensity and position of the amplified products.

[0056] Specifically, according to the previous chip analysis results (gene expression profile data obtained by 22K Human Genome Array chip hybridization in the early stage), one up-regulated gene and one down-regulated gene with significant ratio value differences are selected in the gene expression profile as the target genes for subsequent RT-PCR verification. Then, specific primers are designed based on the sequence information of these target genes. The primers need to ensure high specificity and be able to bind to the specific sites of the target genes. At the same time, the Tm value melting temperature of the primers is similar to optimize the amplification efficiency and ensure the stability of the PCR reaction. In general, the Tm value of the primer should be around 60°C, and the difference in the Tm value of the front and rear primers should not exceed 2°C. After the design is completed, the RT-PCR reaction is carried out:

[0057] By setting the reaction conditions of RT-PCR: the initial denaturation temperature is 95°C and maintained for 30 seconds to ensure that the DNA double strands are completely separated; then, the annealing stage is carried out, the temperature is lowered to 60°C and maintained for 30 seconds to allow the primers to specifically bind to the single-stranded DNA of the target gene; finally, the extension stage is carried out, the temperature is set to 72°C and maintained for 30 seconds, and the target sequence is amplified using Taq DNA polymerase. This cycle is repeated 40 times to obtain sufficient amplified products. After the amplification is completed, the PCR products are separated by agarose gel electrophoresis, 0.7% agarose gel is prepared, and the PCR products are mixed with staining buffer and added to the gel wells. After electrophoresis for a period of time, the gel is placed under ultraviolet light for observation, and the bands of the PCR products are recorded. By comparing the band intensity and position of the PCR products, the expression level of the gene is evaluated. If the band of the up-regulated gene is significantly stronger and the band of the down-regulated gene is weaker, it means that the RT-PCR verification result is consistent with the chip analysis, which further confirms the expression difference of the target gene in ovarian cancer.

[0058] The present invention uses SNP gene chip technology to systematically study the relationship between the polymorphism of XRCC3 and XPC genes and the incidence of ovarian cancer. Through high-throughput gene detection, specific polymorphic sites related to ovarian cancer can be efficiently identified, thereby providing more accurate biomarkers for early screening of ovarian cancer. In addition, the implementation method of the present invention ensures high-quality acquisition of samples and accuracy of data analysis, has the advantages of simple operation, good repeatability and high sensitivity, and contributes to the early diagnosis of ovarian cancer through specific polymorphic sites of XRCC3 and XPC genes.

[0059] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0060] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. An early screening method for ovarian cancer based on XRCC3 and XPC gene analysis, characterized in that: The following steps are involved: Step 1: 40 peripheral blood samples were collected from the experimental group (clinical ovarian cancer patients) and the control group (patients with other benign diseases) to provide basic samples for XRCC3 and XPC gene detection; Step 2: Use a DNA extraction kit to extract whole genome DNA from the collected blood samples, purify it using a DNAClean&Concentrator TM-5 kit to remove inhibitory factors and contaminants, and then measure the DNA concentration using a NanoDrop UV spectrophotometer. Use 0.7% agarose gel for electrophoresis to screen qualified DNA samples; Step 3, preparing a 22K Human Genome Array chip, the chip comprising 21522 oligo DNA probes, wherein the specific probes are used to identify the A4541G, A17893G, T241M sites of the XRCC3 gene and the Ala499Val, Lys939Gln sites of the XPC gene, each probe specifically identifies a human gene transcript, and reversely transcribes the mRNA of the experimental group - clinical ovarian cancer patients and the normal control group - other benign diseases into cDNA, and uses the cDNA as a probe to hybridize with the XRCC3 and XPC specific probes on the chip; Step 4: Quantitative analysis of the extracted DNA samples was performed and standardized to 50 ng / μL. The amount of DNA sample required for chip analysis was 200 ng. 0.1 N NaOH was added to the sample to denature the DNA into a single strand, which then combined with the XRCC3 and XPC gene-specific probes on the chip. Subsequently, the neutralized sample was added to the whole genome amplification reagent and incubated overnight at 37°C to make the total amount of amplified DNA reach 2000-3000 times the initial loading amount. Step 5: The amplified DNA sample is subjected to controlled enzymatic treatment so that the fragmentation process does not damage the integrity of the XRCC3 and XPC genes. After fragmentation, the fragmented DNA is precipitated by adding isopropanol and centrifuged at 4°C to enrich the fragmented DNA, and then dried in air and resuspended in hybridization buffer; Step 6: Hybridize the DNA sample resuspended in hybridization buffer with the prepared chip and place it in a hybridization oven for overnight reaction. At this time, the fragmented DNA effectively combines with the XRCC3 and XPC gene site-specific probes on the chip. The chip contains a total of 610,000 types of microbeads, each of which corresponds to the detection of one SNP site. Step 7: Wash and remove the unhybridized and non-specifically hybridized DNA, retain only the DNA bound to the XRCC3 and XPC gene sites, use the captured DNA to perform a single base extension reaction on the chip, and add a detectable label group on the chip to distinguish the SNP types of different samples in the XRCC3 and XPC genes, and place the chip after the reaction in the XC4 reagent for coating to form a protective layer to stabilize the detection signals of the XRCC3 and XPC genes; Step 8: Scan the chip using a LuxScan 10KA dual-channel laser scanner to excite the fluorescent groups of the single-base extension products on the chip to generate high-resolution images. The obtained data are imported into LuxScan 3.0 image analysis software to analyze the fluorescence signal intensity and ratio between OC tissue and normal tissue, and 12 housekeeping genes are used for balance. In the data analysis, the criteria for determining the differential expression of XRCC3 and XPC genes were set as follows: Standard 1: The ratio of OC tissue to normal tissue must be greater than 2.0 or less than 0.5, and the gene expression change is more than 2 times, reflecting a significant difference; Standard 2: The signal intensity of either OC tissue or normal tissue must be greater than 800, or both must be greater than 200, and the dominant expression data can be repeated in all samples; Step 9. Select one up-regulated gene and one down-regulated gene with significant difference in ratio value in XRCC3 and XPC genes in the gene expression spectrum, and verify them by RT-PCR technology to confirm the difference in expression of XRCC3 and XPC genes in ovarian cancer patients.

2. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In the step three, the reverse transcription reaction of the mRNA adopts CbcScript reverse transcriptase to ensure the reverse transcription efficiency and the integrity of cDNA, by mixing the mRNA collected from the experimental group - clinical ovarian cancer patients and the control group - patients with other benign diseases, and setting the reaction conditions provided in the user manual of CbcScript reverse transcriptase, including appropriate temperature and time. After the reaction is completed, the quality of cDNA is confirmed by agarose gel electrophoresis, and the integrity is verified by comparing the band size of cDNA with the standard marker.

3. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In step 4, the concentration range of the whole genome amplification reagent added is 1-5 μL, and the volume of the reaction system is maintained at 20-50 μL. A thermal cycle program is used during the amplification process, including the following stages: Denaturation stage: Keep at 95°C for 30 seconds to 1 minute to completely separate the DNA double strands; Annealing stage: lower the temperature to 55°C-65°C and keep it for 30 seconds to 1 minute to allow the primer to specifically bind to the target sequence; Extension stage: set the temperature at 72°C and keep it for 30 seconds to 1 minute to amplify the target sequence using Taq DNA polymerase; After amplification, the concentration of the amplified product was measured by a photometer, and the amount of DNA sample obtained met the 200 ng standard required for subsequent chip analysis.

4. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In step 5, the fragmented DNA sample needs to be treated with enzymes, and the enzymes selected include restriction endonucleases or DNA fragmentases. The generated DNA fragments are in the range of 200-500 bp. After DNA precipitation, unbound reagents and enzymes are removed by centrifugation and washing to obtain high-purity fragmented DNA samples, which are then resuspended in an appropriate hybridization buffer.

5. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In step six, the fragmented DNA sample is denatured to convert it into a single-stranded form. At this time, the sequence in the DNA sample is annealed with the site-specific 50-base probes on the chip, and each microbead is connected to a different 50-base sequence.

6. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In the step seven, the chip is placed in the XC4 reagent for coating, so that the surface of the chip is coated with a layer of viscous transparent liquid to form a protective layer outside the chip.

7. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In step eight, the chip that has been treated with hybridization and extension reactions is placed in a scanner, and the fluorescent group of the single-base extension product on the chip is excited by laser. The scanner obtains the fluorescent signal emitted by the fluorescent group and generates high-resolution image data. The obtained image data is directly imported into LuxScan3.0 image analysis software for processing and analysis, and the SNP typing data of each sample is extracted.

8. The method for early screening of ovarian cancer based on XRCC3 and XPC gene analysis according to claim 1, characterized in that: In the step nine, specific primers are designed according to the sequence information of the target gene, and the Tm values ​​of the primers are similar. In the RT-PCR reaction, the initial denaturation temperature is set to 95°C, the annealing temperature is set to 60°C, the extension temperature is set to 72°C, and each cycle is set to 30 seconds. The PCR products are separated by agarose gel electrophoresis to confirm the specificity and size of the amplification, and the expression differences of the up-regulated and down-regulated genes are confirmed by comparing the band intensity and position of the amplified products.

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