SNP (Single Nucleotide Polymorphism) site and application thereof

Abstract

The invention belongs to the technical field of gene diagnosis, and discloses an SNP (Single Nucleotide Polymorphism) site and application thereof. The SNP site is located on an SLC4A4 gene of a No.4 chromosome of a human genome, the SLC4A4 gene has an SNP site rs7689609, and the mutation of the rs7689609 site is related to erectile dysfunction. Wherein when the basic group of the rs7689609 site is T, the subject shows no erectile dysfunction; and when the basic group of the rs7689609 site is C, the subject shows erectile dysfunction. The SNP site related to the erectile dysfunction is screened out and is expected to be used in the fields of erectile dysfunction diagnosis and the like, the diagnosis efficiency is improved, and the diagnosis time is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of gene diagnosis and relates to a SNP site and application thereof. Background Art

[0002] Genome-Wide Association Studies (GWAS) is a multi-center, large-sample, and repeatedly validated study of the association between genes and diseases at the whole genome level. It searches for genetic factors associated with complex diseases by typing large-scale population DNA samples with high-density whole-genome genetic markers such as SNPs, thereby comprehensively revealing genetic genes related to the occurrence, development, and treatment of diseases.

[0003] SNPs screened by GWAS have achieved many gratifying results in the field of disease diagnosis. This technology compares the SNP sites of alleles in healthy and diseased populations to explore the relationship between these SNP sites and the occurrence and development of diseases, providing a powerful means for the diagnosis of a variety of diseases, such as tumors, diabetes, hypertension, macular degeneration and other diseases. However, due to the extremely rich SNP sites involved in the whole genome, the comprehensive discovery of disease-related SNPs is still facing great challenges, and the number of SNP sites and the prediction accuracy need to be improved.

[0004] Erectile dysfunction (ED) is a common disease in men, and its pathogenesis is complex. Currently, research on SNP sites related to ED is still scarce. Therefore, improving the research level of GWAS, expanding its practical clinical application scope, and promoting the development of individualized and accurate diagnosis of ED are technical issues that need to be urgently addressed in existing ED diagnostic technologies. Summary of the invention

[0005] In order to further enrich the existing technical means for ED diagnosis, promote the accurate diagnosis of ED, and eliminate or reduce the adverse effects of ED on patients, the present invention provides the following technical solutions.

[0006] The present invention first provides a SNP site, wherein the SNP site is located in the SLC4A4 gene on chromosome 4 of the human genome, the SLC4A4 gene has a SNP site rs7689609, and a mutation in the rs7689609 site is associated with erectile dysfunction.

[0007] Furthermore, when the base at the rs7689609 site is T, the subject exhibits no erectile dysfunction; and when the base at the rs7689609 site is C, the subject exhibits erectile dysfunction.

[0008] Furthermore, the upstream primer for specific detection of the rs7689609 site is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2.

[0009] In addition, the present invention also seeks to protect the use of the above-mentioned SNP site in the preparation of gene chips, molecular probes or kits.

[0010] Finally, the present invention also claims protection for a gene chip, molecular probe or kit, which is used to detect the gene mutation of the above-mentioned SNP site and perform genotyping according to the detection results. When the base of the SNP site is T, the subject shows no erectile dysfunction, and when the base of the SNP site is C, the subject shows erectile dysfunction.

[0011] Furthermore, for the above gene chip, molecular probe or kit, the upstream primer for specifically detecting the SNP site is shown as SEQ ID NO: 1, and the downstream primer is shown as SEQ ID NO: 2.

[0012] Compared with the prior art, the present invention "a SNP site and its application" has the following beneficial effects:

[0013] The present invention clarifies the relationship between the gene mutation at the rs7689609 site of the SLC4A4 gene and the phenotype: when the base of rs7689609 is T, the subject exhibits no erectile dysfunction, and when the base of rs7689609 is C, the subject exhibits erectile dysfunction.

[0014] In addition, the present invention also provides a specific primer pair for detecting the above SNP site, the upstream primer of the primer pair is shown as SEQ ID NO: 1, and the downstream primer is shown as SEQ ID NO: 2. The primer pair can be used for genetic diagnosis of ED.

[0015] Based on the above applications, technicians in this field can achieve rapid diagnosis of ED, thereby shortening traditional diagnosis time and improving diagnosis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the original data images after extraction from different samples. Figure 1 The different letters in the table are sample numbers.

[0017] Figure 2 It is the Manhattan plot of the SNP site described in the present invention. Figure 2 In the figure, the vertical axis is the log 10 represents the association P value; the horizontal axis represents the position of the SNP site on the chromosome. DETAILED DESCRIPTION

[0018] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all 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.

[0019] The main reagents and consumables involved in the examples are as follows.

[0020] Whole genome DNA extraction kit: Thermo Fisher Biotechnology Company, USA;

[0021] Agarose (Biowest Agarose): Axygen, Shanghai Biotech Reagent, USA;

[0022] TBE (Tris-boracic acid) electrophoresis buffer: Tris base 53.88g, EDTA powder 2.93g, boric acid (HgBO3) powder 27.5g, dissolve in water, adjust pH to 8.3 and make up to 1L (this is 5× storage solution); dilute to 0.5× with water for electrophoresis.

[0023] 6× loading buffer: Bio-Technology Co., Ltd.;

[0024] Dured nucleic acid dye: Beijing Fanbo Biological Company;

[0025] 1×TE solution: Shanghai Biochemical Reagents, Axygen, USA;

[0026] DNA standard buffer (FG3): Axygen, USA, Shanghai Biotechnology Reagents

[0027] 1×TE solution: Shanghai Bioengineering Reagents, Axygen, USA

[0028] Axiom2.0 Denat Soln 10×、Axiom 2.0Neutral Soln、Axiom2.0 Amp Soln、Axiom Water、Axiom2.0 Amp Enzyme、Denaturation Master Mix(DS)、AmplificationMaster Mix(AM)、Axiom 10×Frag Buffer、Axiom Frag Diluent、Axiom Frag Enzyme、Axiom Frag Rxn Stop、Precip Soln, Fragmentation Master Mix(FM), PrecipitationMaster Mix(PM), Axiom Hyb Buffer, Axiom Hyb Soln, Axiom Resusp Buffer, Hybridization Master Mix, Axiom Ligate Enzyme, Axiom Wash A, Axiom Ligate Soln2, Axiom Hold Buffer: Thermo Fisher Biotechnology Company of the United States;

[0029] Centrifuge: Eppendorf Life Sciences, Germany;

[0030] Vortex oscillator: Beijing Tektronix Instrument Manufacturing Co., Ltd.;

[0031] Medical 37℃ constant temperature box, electrophoresis apparatus and matching electrophoresis tank, gel imaging system, full-wavelength UV / visible light scanning spectrophotometer (NanoDrop-1000 / NanoDrop-2000), preheated hybridization oven, GeneTitan chip platform, PCR instrument: Thermo Fisher Biotechnology Company, USA;

[0032] Refrigerator, microwave oven: Qingdao Haier Home Appliances Co., Ltd.;

[0033] Electronic scale: Changzhou Hongheng Electronic Instrument Factory.

[0034] Example 1

[0035] This example describes sample collection and genomic DNA extraction.

[0036] 1. Whole Blood Sample Collection

[0037] About 5 mL of peripheral venous blood was collected from patients with erectile dysfunction (ED) and patients in the negative control group using EDTA anticoagulation tubes, and the collected fresh blood samples were centrifuged at 3000 rpm for 10 min to separate blood cells and stored at -20°C.

[0038] 2. Genomic DNA Extraction

[0039] 1) Use a pipette to transfer 2 mL of blood cells into a 15 mL centrifuge tube, add 10 mL of buffer NL reagent, and let stand at room temperature for 10 minutes to allow the blood cells to fully lyse.

[0040] 2) After lysis, the mixture was centrifuged at room temperature (25°C, 3000g, 10 min).

[0041] 3) Discard the supernatant, use filter paper to absorb the remaining supernatant, and place the centrifuge tube upside down on the filter paper for about 5 minutes to allow the supernatant to be fully absorbed.

[0042] 4) Add 2 mL of the prepared mixture of buffer XL and buffer OB to the centrifuge tube to lyse the cell nuclei (the mixture needs to be used within 10 minutes after preparation). Place the centrifuge tube on a vortex oscillator and oscillate at 200× for 10 to 15 seconds to fully lyse the cell nuclei.

[0043] 5) Place the centrifuge tube in a 65°C water bath and incubate for 30 minutes until the color of the liquid in the centrifuge tube changes from red to olive green, then take it out.

[0044] 6) Add 2 mL of isopropanol to the centrifuge tube to precipitate the DNA. Then gently shake the centrifuge tube 20 to 30 times until the liquid precipitates. An obvious white precipitate indicates that the DNA has been precipitated well.

[0045] 7) Centrifuge the centrifuge tube at room temperature (25°C, 3000g, 5min), discard the supernatant, use filter paper to absorb a small amount of residual supernatant and invert the centrifuge tube on the filter paper for 3-5min to allow the supernatant to be fully absorbed without residue.

[0046] 8) Add 1 mL of 70% alcohol solution and vortex on a vortex oscillator at 150× for 10 seconds to wash the DNA.

[0047] 9) Centrifuge at room temperature (25°C, 2000g, 3min) and discard the supernatant. Pay attention to the position of the precipitate to prevent DNA loss. If the DNA is found to be red, repeat the alcohol washing and centrifugation operation to fully wash the DNA.

[0048] 10) Place the centrifuge tube upside down on clean filter paper and let it stand for 10 to 15 minutes to allow the alcohol to evaporate.

[0049] 11) Add 200-500 μL buffer EB solution and place in a 65°C water bath for 1 hour to dissolve the DNA.

[0050] 12) Store the dissolved DNA at -20°C.

[0051] Example 2

[0052] This example describes genomic DNA electrophoresis, concentration detection and normalization.

[0053] 1. Genomic DNA Electrophoresis

[0054] 1) Weigh agarose to prepare 1% agarose gel, add TBE working solution of equal volume to agarose gel, and shake well.

[0055] 2) Heat in a microwave oven for 1 to 2 minutes until the agarose is completely dissolved.

[0056] 3) After the solution has cooled to about 60°C, add Dured nucleic acid dye (5 μL per 50 mL agarose solution) to a working concentration of 1× and mix thoroughly.

[0057] 4) Pour the agarose gel into the gel mold and place the comb so that it is 0.5 to 1.0 mm away from the bottom of the gel mold to form a sample loading hole. At the same time, check to ensure that there are no bubbles in the gel to avoid affecting the electrophoresis results.

[0058] 5) Let stand at room temperature for 30 to 40 minutes. After the gel cools and solidifies, remove the comb and transfer the gel to the electrophoresis tank for later use.

[0059] 6) Add enough electrophoresis buffer to cover the gel by about 1 to 2 mm.

[0060] 7) Evenly mix the DNA sample and the loading buffer, and accurately add 5 μL of the mixture into the sample well using a pipette.

[0061] 8) Check the positive and negative poles of the electrophoresis tank and the lid to ensure that the lid is powered and properly connected. Perform electrophoresis at 120V for 45 to 60 minutes until bromophenol blue migrates to about 2 / 3 of the gel.

[0062] 9) Take out the gel, examine the gel under UV light and scan the image.

[0063] 2. DNA concentration detection and standardization

[0064] DNA concentration detection: A was measured using a NanoDrop-1000 spectrophotometer. 260 / A 280 (protein and phenol residue index) and A 260 / A 230 (Indicator of residual inorganic salts and small molecule impurities). For samples undergoing whole genome genotyping, A 260 / A 280 The ratio should be controlled between 1.7 and 2.0. 260 / A 230 The ratio should be between 1.5 and 2.3.

[0065] Concentration standardization: Samples that meet the quality standards are diluted to the standard experimental concentration. The concentration for DNA experiments should be above 40ng / μL, and the total amount of DNA should be above 100ng.

[0066] Example 3

[0067] This example describes genotyping of genomic DNA.

[0068] Axiom gene chip typing technology uses enzyme-mediated, single-base sequencing steps to ensure high repeatability of experimental typing results and full automation of the process. The entire operation process of the Axiom chip experiment is simple and direct. The entire experiment requires 200ng of starting DNA. After whole genome amplification, the product is randomly fragmented into fragments between 25 and 125bp. These fragments are purified and resuspended, and hybridized with the Axiom human whole genome microplate chip. After the hybridization process is completed, rigorous washing is performed, and non-specific background will be removed while specific binding is retained. Each SNP site is identified by a multi-color connection reaction occurring on the surface of the chip. When the connection reaction is completed, the chip will complete the staining and washing steps on the GeneTitan multi-channel automated chip workstation, and finally scan and output the results. The specific experimental process is as follows.

[0069] 1. DNA Amplification

[0070] 1) Take the genomic DNA out of the refrigerator, wait for it to return to room temperature, and then dilute it with sterile water or TE buffer to prepare a genomic DNA sample with a concentration of 10 ng / μL.

[0071] 2) Add 20 μL of the sample prepared above to each well of a clean 96-well plate.

[0072] 3) Prepare the denaturation buffer master solution Denaturation Master Mix (DS): Axiom 2.0 Denat Soln 10×, 2μL and Axiom Water, 18μL, mixed to 20μL DS. Dispense the prepared Denaturation Master Mix (DS) into 24-channel tubes at 100μL / well, and add 20μL of prepared DS to each sample well. Add the reagent to the bottom of the sample tube without blowing or aspirating, seal the film, shake and mix, place for 10 minutes, and then centrifuge at room temperature (1000-2000rpm, 1-2min); after incubation at room temperature for 10 minutes, immediately start the next step.

[0073] 4) Pour the neutralization and denaturation buffer Axiom 2.0 Neutral Soln (NS) into a clean reagent tank, add 130 μL NS to each sample, seal the membrane, oscillate, and centrifuge (same conditions as above), and immediately start the next step after completion.

[0074] 5) Prepare Amplification Master Mix (AM): Mix Axiom2.0 Amp Soln and Axiom2.0 Amp Enzyme at a volume ratio of 225:5, vortex thoroughly and turn the centrifuge tube upside down to ensure thorough mixing.

[0075] 6) Add 230 μL of the AM prepared above to each sample well, place the pipette tip against the tube wall, and let it flow in slowly without blowing or aspirating.

[0076] 7) Wipe the liquid on the plate dry, seal the film and press tightly, shake twice, and centrifuge at 1000 rpm for 1 min at room temperature.

[0077] 8) Place the sample plate with AM added in a hybridization oven at 37°C and incubate for 23 hours.

[0078] 2. DNA fragmentation and precipitation after amplification

[0079] 1) After amplification, the sample plate was taken out from the hybridization oven and incubated in a 65°C hybridization oven for 20 min. The sample plate was then taken out and incubated in a 37°C hybridization oven for 45 min.

[0080] 2) After the incubation, take out and add 57 μL of fragmentation buffer master mix (FM) to each well, seal the sealing film, shake and mix, and leave for 30 minutes. The preparation method of the FM is: Axiom 10×Frag Buffer, Axiom Frag Diluent, and Axiom Frag Enzyme are mixed in a ratio of 45.7:10.3:1. The FM is prepared in advance and used within 5 minutes after preparation.

[0081] 3) After the sample plate is placed, centrifuge it at room temperature and return it to the 37°C hybridization oven for 30 minutes.

[0082] 4) Immediately after completion, add 19 μL of Axiom Frag Rxn Stop reagent to each well, seal with sealing film, oscillate to mix, and centrifuge at 1000-2000 rpm for 1-2 minutes at room temperature to stop the fragmentation reaction and precipitate DNA fragments. The Axiom Frag Rxn Stop reagent should be taken out of the refrigerator in advance and restored to room temperature for use.

[0083] 5) Add 240 μL Precipitation Master Mix (PM) to each well, seal with sealing film, oscillate to mix, and centrifuge at 2000 rpm for 2 minutes at room temperature to promote DNA precipitation. The PM is prepared by mixing Axiom Precip Soln 1 and Axiom Precip Soln 2 at a volume ratio of 238:2. PM should be prepared in advance.

[0084] 6) Add 600 μL of isopropanol to each well, mix thoroughly by blowing and sucking several times with a pipette, seal the well with sealing film, and place the sample in a -20°C refrigerator for 16 to 24 hours.

[0085] 3. Drying, resuspension and quality control of fragmented DNA

[0086] 1) DNA drying: After the sample is placed, take out the sample plate and place it in a pre-cooled 4°C centrifuge. Centrifuge at 3200×g for 40 minutes. Discard the supernatant, turn the sample plate upside down on absorbent paper for 5 minutes, and absorb the residual liquid in the tube. Turn the sample plate over and place it at 37°C for drying for 10-20 minutes.

[0087] 2) Resuspension: Add 35 μL of Resusp Buffer to each sample well and dissolve on an oscillator for 20 minutes.

[0088] 3) Transfer all dissolved samples to a new 96-well plate, add 80 μL of hybridization solution to each well, seal the plate, shake, and centrifuge. The hybridization solution is prepared by mixing Axiom Hyb Buffer, Axiom Hyb Soln 1, and Axiom Hyb Soln 2 in a volume ratio of 70.5:0.5:9.

[0089] 4) Quality control: Take 33 μL of sterile water and 3 μL of the sample to be hybridized (i.e., the liquid in step 2)) to a new 96-well plate, seal the film, shake and centrifuge, and record the 96-well plate as the "QC Diln" plate. Take 10 μL of the "QC Diln" plate sample and 90 μL of sterile water to a new 96-well plate, record it as the "OD" plate. Take 3 μL of the "QC Diln" plate sample for electrophoresis, with a gel concentration of 4%; in addition, use an ELISA reader to measure the concentration of the "OD" plate sample.

[0090] The size, integrity and purity of the DNA fragments are determined based on the electrophoresis results. The electrophoresis pattern of a qualified sample should show clear bands without obvious degradation or contamination.

[0091] 4. DNA Denaturation Hybridization

[0092] 1) Start the GTGS software, create a project and sample plate in the specified folder, and enter the sample information to be hybridized.

[0093] 2) Chip preheating: Chip preheating is usually performed in a hybridizer, and the preheating temperature and duration are set according to the experimental manual. After preheating, open the package and scan the chip barcode into the GeneTitan MC system to facilitate system recognition and record chip information.

[0094] 3) After confirming that the GeneTitan system can hybridize the new chip, start the denaturation procedure. Denaturation procedure: 95℃10min; 48℃3min; 48℃ insulation (if the sample to be hybridized is stored at -20℃, it needs to be fully thawed at room temperature in advance).

[0095] 4) Take 105 μL of denatured sample into the hybridization tray, which is used to load the denatured sample and transfer it to the chip, taking care not to have bubbles. The hybridization tray and denatured sample are placed on a metal block preheated at 48°C.

[0096] 5) According to the prompts of the GeneTitan MC system, place the hybridization tray and chip (remove the cover and keep the bracket) in the designated position. After the system assembles the hybridization tray and chip, the system ejects the chip and checks whether it is assembled tightly and whether there are bubbles.

[0097] 6) After checking that everything is correct, start hybridization, and the hybridization time is 23.5 to 24 hours.

[0098] 7) Record the position of the chip barcode on the GeneTitan hybridizer.

[0099] 5. Chip cleaning, staining, and scanning

[0100] 1) Preparation of Stain 1 Master Mix: Mix Axiom Wash A, Axiom Stain Buffer, Axiom Stain 1-A, and Axiom Stain 1-B in a ratio of 201.6:4.2:2.1:2.1.

[0101] 2) Preparation of Stain 2 Master Mix: Mix Axiom Wash A, Axiom Stain Buffer, Axiom Stain 2-A, and Axiom Stain 2-B in a ratio of 100.8:1.1:1.05:1.05.

[0102] 3) Preparation of Stabilization Master Mix: Mix Axiom Water, Axiom Stabilize Diluent, and Axiom Stabilize Soln in a ratio of 93.19:10.5:1.31.

[0103] 4) Ligation Master Mix 1: Stage 1 (Lig-M1): Mix Axiom Ligate Buffer, Axiom Ligate Soln 1, and Axiom Ligate Soln 2 in a ratio of 66.15:13.12:3.15. Mix Lig-M1 thoroughly with an oscillator before preparing Lig Mix.

[0104] 5) Ligation Master Mix: Stage 2 (Lig Mix): Mix Lig-M1, Axiom Prob Mix 1, Axiom Prob Mix 2, and Axiom Ligate Enzyme in a ratio of 82.42:10.5:10.5:1.58. Invert the Lig-Mix several times to ensure thorough mixing.

[0105] 6) Stain 1 Master Mix was dispensed into 2 portions for each sample, 105 μL each; Stain 2 Master Mix and Stabilization Master Mix were dispensed into 1 portion for each sample, 105 μL each.

[0106] 7) Add Holding Buffer to the scanning plate, 150 μL per well.

[0107] 8) Label each board on the side with the affy label.

[0108] 9) Add various staining reagents in sequence according to the software prompts.

[0109] 10) Place the plate and lid into the GeneTitan MC, making sure the missing corner is aligned and positioned correctly.

[0110] 11) When placing the scanning disc into the GeneTitan MC, remove the base.

[0111] 12) After the scan is completed, remove the chip from the system, place the chip on a stand, seal it, and store it at 4°C.

[0112] 6. Experimental Results

[0113] Figure 1 Original images of genome sequencing after DNA extraction from different samples.

[0114] Figure 2 It is the Manhattan plot of the SNP site described in the present invention. Figure 2 In the figure, the vertical axis is the log 10 represents the association P value; the horizontal axis represents the position of the SNP site on the chromosome. Figure 2 It can be seen that there are three independent SNP sites in the SLC4A4 gene on chromosome 4, all of which are significantly correlated with ED, including rs7689609, indicating that the rs7689609 site of the SLC4A4 gene is highly correlated with the occurrence of ED and can be used to determine the occurrence of ED.

[0115] In summary, the allele frequencies of 533,568 SNP loci were compared between the ED population and the control population using a genome-wide association study (GWAS), and all the P values ​​associated with the occurrence of ED were less than 5×10 -5 The SNP sites of chromosome 4 SLC4A4 gene rs7689609 were extracted. Among them, the SNP site of chromosome 4 SLC4A4 gene rs7689609 is a SNP site highly correlated with ED (P=8.853e-07). When the base of this site mutates from T to C, the subject exhibits erectile dysfunction. This site is conducive to the screening of ED and is also a good candidate gene for further searching for genes related to ED. The present invention provides a reference for understanding the relationship between SLC4A4 gene mutation and ED and its application.

[0116] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A SNP site, characterized in that: The SNP site is located in the SLC4A4 gene on chromosome 4 of the human genome. The SLC4A4 gene has a SNP site rs7689609, and a mutation in the rs7689609 site is associated with erectile dysfunction.

2. The SNP site according to claim 1, characterized in that: When the base at the rs7689609 site is T, the subject shows no erectile dysfunction; when the base at the rs7689609 site is C, the subject shows erectile dysfunction.

3. The SNP site according to claim 2, characterized in that: The upstream primer for specific detection of the rs7689609 site is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO:

2.

4. Use of the SNP site according to claim 1 in preparing a gene chip, a molecular probe or a kit.

5. A gene chip, molecular probe or kit, characterized in that: The gene chip, molecular probe or kit is used to detect the gene mutation of the SNP site of claim 1, and perform genotyping according to the detection results; When the base at the SNP site is T, the subject exhibits no erectile dysfunction; when the base at the SNP site is C, the subject exhibits erectile dysfunction.

6. The gene chip, molecular probe or kit according to claim 5, characterized in that: The upstream primer for specifically detecting the SNP site is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2.

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