Construction method of gene mutation library detected by nucleic acid mass spectrometry

Abstract

The invention discloses a construction method of a gene mutation library detected by nucleic acid mass spectrometry, which comprises the following steps: searching a gene sequence related to tumor radiotherapeutic toxicity, collecting and processing a sample, extracting and purifying nucleic acid, determining a gene segment related to the radiotherapeutic toxicity, amplifying the gene segment related to the radiotherapeutic toxicity, and constructing the gene mutation library by using the nucleic acid mass spectrometry. A mutation site is introduced into a gene segment related to radiotherapeutic toxicity, a long nucleic acid segment is subjected to fragmentation treatment, a carrier is connected, a connection product is converted and amplified, and library quality is controlled. By constructing the gene mutation library for nucleic acid mass spectrometry detection, gene functions can be systematically studied on the gene level, specific effects of genes in normal physiological and pathological processes are determined, gene variation conditions in disease states are simulated, disease progression is predicted, resources are provided for discovery of drug targets, and the method has a wide application prospect. The medicine curative effect and the medicine reaction mechanism are evaluated, precise medical treatment is achieved, and convenience is provided for malignant tumor treatment work.

Description

Technical Field

[0001] The present invention relates to the field of gene sequencing, and in particular to a method for constructing a gene mutation library using nucleic acid mass spectrometry detection. Background Art

[0002] Tumors are new growths caused by abnormal proliferation of cells in local tissues under the influence of various tumorigenic factors. Tumors can be divided into benign tumors and malignant tumors in nature. Malignant tumors grow rapidly and in an invasive manner. They can invade surrounding tissues and organs and can also metastasize to other parts of the body through the blood or lymphatic system. Malignant tumors have become one of the major public health issues that seriously threaten the health of the Chinese population. Tumor radiotherapy is a local treatment method that uses radiation to treat tumors. Normal tissue cells and tumor cells have different sensitivities to radiation. Tumor radiotherapy uses this difference to minimize damage to normal tissues while maximizing the killing of tumor cells.

[0003] During the process of radiotherapy for tumors, the rays produced by radiotherapy may destroy the normal structure of genes, causing various mutation forms such as point mutations, deletions, and insertions. Mutated toxic genes may reduce the repair ability of cells, leading to more serious cell death or functional disorders, or they may enable cells to gain stronger repair ability, thereby reducing the efficacy of radiotherapy and increasing drug resistance. The uncertainty after toxic gene mutations greatly increases the difficulty of treatment. Therefore, it is necessary to construct a gene mutation library for nucleic acid mass spectrometry detection to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a gene mutation library detected by nucleic acid mass spectrometry to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for constructing a gene mutation library detected by nucleic acid mass spectrometry, comprising the following steps: Step 1: Check the gene sequences related to tumor radiotherapy toxicity; Step 2: Collect and process samples; Step 3: Extract and purify nucleic acids to identify gene fragments associated with radiotherapy toxicity; Step 4: Amplify gene fragments associated with radiation therapy toxicity; Step 5: Introducing mutation sites into gene segments associated with radiotherapy toxicity; Step 6: Fragmentation of longer nucleic acid fragments; Step 7: Connect the carrier; Step 8: Transform the ligation product and amplify it; Step 9: Control library quality.

[0006] Preferably, the samples collected in step 2 include blood samples and tissue samples, the tissue samples are obtained by surgical resection and puncture biopsy, the blood samples need to avoid hemolysis, and the processing steps of the tissue samples include cutting the tissue samples into small pieces and adding lysis solution and proteinase K, and the lysis solution contains sodium dodecyl sulfate.

[0007] Preferably, the method for extracting and purifying nucleic acid in step three is a silica gel membrane adsorption method, wherein the salt ion concentration range of the high salt concentration environment in the silica gel membrane adsorption method is 4-6 mol / L, and guanidine salt is used.

[0008] Preferably, the amplification method in step 4 is polymerase chain reaction, comprising the following steps: S1: Heat the temperature to 94-98℃ to unwind the double-stranded DNA into two single-stranded DNAs; S2: Lower the temperature to 50-65°C and use primers to bind to the complementary sequence on the single-stranded DNA template; S3: At a temperature of 72°C, with the help of DNA polymerase and single-stranded DNA as a template, starting from the 3' end of the primer, free deoxynucleotides are added to the primer and then extended to form a new DNA chain according to the principle of base complementary pairing.

[0009] Preferably, the method used in step five is based on the overlap extension method of polymerase chain reaction, and the mutation is introduced into the gene fragment related to the toxicity of radiotherapy with the help of primers containing the mutation site.

[0010] Preferably, the method adopted in step six is ​​ultrasonic crushing, and the frequency of ultrasonic waves in the ultrasonic crushing is 30-50kHz.

[0011] Preferably, connecting the vector in step seven refers to connecting the gene fragment associated with radiotherapy toxicity to the vector.

[0012] Preferably, the ligation product in step eight needs to be inserted into a host cell for transformation.

[0013] Preferably, the quality of the library in step nine includes the concentration and purity of the library.

[0014] Technical effects and advantages of the present invention: The present invention can systematically study gene function at the gene level by constructing a gene mutation library for nucleic acid mass spectrometry detection, determine the specific role of genes in normal physiological and pathological processes, simulate gene variation under disease conditions, predict disease progression, provide resources for the discovery of drug targets, evaluate drug efficacy and drug response mechanisms, realize precision medicine, and greatly facilitate the treatment of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0016] 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.

[0017] The present invention provides Figure 1 A method for constructing a gene mutation library for nucleic acid mass spectrometry detection is shown, comprising the following steps: Step 1: Look up gene sequences related to tumor radiotherapy toxicity. The data can be obtained from literature, the Internet, and databases. The gene sequences related to tumor radiotherapy toxicity found are sorted and arranged for easy comparison; Step 2: Collect and process samples. The collected samples include blood samples and tissue samples. Blood samples can be obtained from tumor patients who have received radiotherapy using a blood drawer. Hemolysis of blood samples must be avoided. Tissue samples can be obtained from tumor patients who have received radiotherapy through surgical resection and puncture biopsy. Optional tissue samples include tumor tissue and normal tissue margins. The processing steps of tissue samples include cutting tissue samples into small pieces and adding lysis solution and proteinase K. Cutting tissue samples into small pieces is to increase the contact area between tissue and lysis solution. Lysis solution contains sodium dodecyl sulfate, which can destroy cell membranes and nuclear membranes and release nucleic acids. Proteinase K can degrade proteins and remove proteins bound to nucleic acids. Step 3: Extract and purify nucleic acids, and determine the gene fragments related to radiotherapy toxicity. The method for extracting and purifying nucleic acids is the silica gel membrane adsorption method. In a high-salt environment, the lysate after lysing the tissue sample is added to a centrifugal column containing a silica gel membrane. In a high-salt environment, nucleic acids will be adsorbed to the silica gel membrane, while impurities such as proteins and polysaccharides cannot be adsorbed to the silica gel membrane. Therefore, impurities such as proteins and polysaccharides can be removed by washing, and then the nucleic acids can be eluted from the silica gel membrane by a low-salt buffer. Finally, ribonucleases can be used to specifically hydrolyze RNA. Ribonucleases can recognize the phosphodiester bonds in RNA molecules and cut them, thereby degrading RNA into small fragments of nucleotides. Step 4: Amplify the gene fragment related to radiation therapy toxicity. The amplification method is polymerase chain reaction, which includes the following steps: S1: Heat the temperature to 94-98℃ to unwind the double-stranded DNA into two single-stranded DNAs; S2: Lower the temperature to 50-65°C and use primers to bind to the complementary sequence on the single-stranded DNA template. The length of the primers is 18-25 dp. S3: At 72°C, with the help of DNA polymerase, using single-stranded DNA as a template, starting from the 3' end of the primer, free deoxynucleotides are added to the primer and extended to form a new DNA chain according to the principle of base complementary pairing; Step 5: Introducing mutation sites into gene segments associated with radiotherapy toxicity, using an overlap extension method based on polymerase chain reaction, by designing primers containing mutation sites, and introducing mutations into gene segments associated with radiotherapy toxicity through multiple rounds of polymerase chain reaction; Step 6: Fragment the longer nucleic acid fragments using ultrasonic fragmentation, which uses high-frequency sound waves emitted by an ultrasonic instrument to generate vibrations in the liquid medium, causing the nucleic acid solution to form tiny cavitation bubbles. These bubbles will generate strong shear forces when they burst instantly, thereby breaking the nucleic acid fragments. The frequency of ultrasonic waves in the ultrasonic fragmentation method is 30-50kHz, and the size of the nucleic acid fragments after cutting can be controlled by controlling the intensity and time of the ultrasonic waves; Step 7: Connect the vector. Use DNA ligase in the presence of a buffer at 16°C to connect the gene sequence associated with radioactive therapy toxicity to the vector. The vectors that can be used include plasmids and bacteriophages. Step 8: transforming and amplifying the ligated product, introducing the ligated product into E. coli for transformation, culturing and amplifying the clones successfully transformed by antibiotic screening, thereby obtaining a library containing the target gene; Step 9: Use a spectrophotometer to detect the concentration and purity of the library to ensure the quality of the library.

[0018] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a gene mutation library for nucleic acid mass spectrometry detection, characterized in that: The following steps are involved: ； Step 1: Check the gene sequences related to tumor radiotherapy toxicity; Step 2: Collect and process samples; Step 3: Extract and purify nucleic acids to identify gene fragments associated with radiotherapy toxicity; Step 4: Amplify gene fragments associated with radiation therapy toxicity; Step 5: Introducing mutation sites into gene segments associated with radiotherapy toxicity; Step 6: Fragmentation of longer nucleic acid fragments; Step 7: Connect the carrier; Step 8: Transform the ligation product and amplify it; Step 9: Control library quality.

2. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The samples collected in step 2 include blood samples and tissue samples. The tissue samples are obtained by surgical resection and puncture biopsy. Hemolysis of the blood samples needs to be avoided. The processing steps of the tissue samples include cutting the tissue samples into small pieces and adding lysis solution and proteinase K. The lysis solution contains sodium dodecyl sulfate.

3. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The method for extracting and purifying nucleic acid in step three is a silica gel membrane adsorption method. The salt ion concentration range of the high salt concentration environment in the silica gel membrane adsorption method is 4-6 mol / L, and guanidine salt is used.

4. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The amplification method in step 4 is polymerase chain reaction. The following steps are involved: S1: Heat the temperature to 94-98℃ to unwind the double-stranded DNA into two single-stranded DNAs; S2: Lower the temperature to 50-65°C and use primers to bind to the complementary sequence on the single-stranded DNA template; S3: At a temperature of 72°C, with the help of DNA polymerase and single-stranded DNA as a template, starting from the 3' end of the primer, free deoxynucleotides are added to the primer and then extended to form a new DNA chain according to the principle of base complementary pairing.

5. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The method adopted in step five is based on the overlap extension method of polymerase chain reaction, and the mutation is introduced into the gene fragment related to the toxicity of radiotherapy by means of primers containing mutation sites.

6. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The method adopted in step six is ​​ultrasonic crushing, in which the frequency of ultrasonic waves is 30-50kHz.

7. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: Connecting the vector in step seven refers to connecting the gene fragment associated with radiotherapy toxicity to the vector.

8. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The ligation product in step eight needs to be inserted into the host cell for transformation.

9. The method for constructing a gene mutation library detected by nucleic acid mass spectrometry according to claim 1, characterized in that: The quality of the library in step nine includes the concentration and purity of the library.

Images