SNP (Single Nucleotide Polymorphism) gene detection interpretation method based on pyrosequencing

Abstract

The invention discloses an interpretation method for SNP (Single Nucleotide Polymorphism) gene detection based on pyrosequencing, and the interpretation method is used for interpreting a pyrosequencing result of an rs1799762 site of a PAI-1 gene, and a sequence to be detected is detected according to a TACACG base sequence during sequencing. Compared with an existing sequencing reading method, the reading method has the advantages that the PAI-1 gene polymorphism can be visually judged through peak patterns, misjudgment among three genotypes is reduced, a qualitative judgment result is visual and accurate, the false positive rate of a pyrosequencing result is greatly reduced, the application range is widened, the labor cost is reduced, and the method is suitable for popularization and application. And a thought is provided for finding more similar gene loci for qualitative judgment.

Description

Technical Field

[0001] The present invention relates to a method for interpreting SNP gene detection based on pyrophosphate sequencing, and belongs to the field of gene sequencing. Background Art

[0002] Pyrosequencing is a novel enzyme-linked cascade sequencing technology suitable for sequencing and analyzing known short sequences. Its reproducibility and accuracy are comparable to Sanger DNA sequencing, while significantly faster. Pyrosequencing products are capable of sequencing and analyzing large numbers of samples simultaneously, providing an ideal platform for high-throughput, low-cost, timely, rapid, and intuitive single nucleotide polymorphism (SNP) research and clinical testing. Existing pyrosequencing methods only allow the addition of one of four dNTPs (dATP, dTTP, dCTP, and dGTP) in each sequencing reaction. Once the dNTP matches the template to be tested, the polymerase catalyzes its incorporation into the primer strand, releasing an equimolar number of pyrophosphate groups (PPi). Sulfurylase catalyzes the formation of equimolar ATP from APS and PPi. ATP drives the luciferase-mediated conversion of luciferin to oxy-luciferin, which emits a visible light signal proportional to the amount of ATP. The light signal is detected by a CCD camera and is reflected as a peak by the Pyrogram. The height of the peak (light signal intensity) is proportional to the number of nucleotides incorporated in the reaction. ATP and unincorporated dNTPs are degraded by diphosphatases, quenching the light signal and regenerating the reaction system. The next dNTP can then be added. As the above process continues in a cycle, complementary DNA chains are synthesized, and the DNA sequence is determined by the Pyrogram signal peak. The presence or absence of the signal peak determines the type of base, and the peak height determines the number of bases.

[0003] In current applications of pyrosequencing, the signal peak ratio is used to determine the number of bases. In actual operation, when encountering fragments with multiple repetitive single bases, inaccurate peak heights may occur, especially for base-deficient gene sites. The PAI-1 gene is a typical example of this.

[0004] PAI-1, short for plasminogen activator inhibitor-1, is the primary substance in the bloodstream that inhibits fibrinolytic activity. It is the primary inhibitor of tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) and a rapid inhibitor of t-PA. The dynamic balance between t-PA and PAI-1 plays a crucial role in maintaining homeostasis in the plasma fibrinolytic system (also known as the fibrinolytic system). The promoter region of the PAI-1 gene contains a G insertion (or deletion) 675 bp upstream of the transcription start site, resulting in a 4G / 5G polymorphism that is closely correlated with plasma PAI-1 activity. The 4G allele elevates plasma PAI-1 levels, disrupting the coagulation-fibrinolytic system, leading to insufficient blood supply to the embryo and potentially causing spontaneous abortion. Studies have found that PAI-1 levels are significantly higher in women with pregnancy complications than in healthy women. PAI-1 is thought to be associated with trophoblast infiltration of the vascular endothelium and fibrin deposition in early pregnancy, contributing to adverse early pregnancy outcomes such as recurrent miscarriage, stillbirth, and premature birth. Clueck et al. studied hereditary hypofibrinolysis in Jewish women with adverse pregnancy outcomes and concluded that this condition, modulated by the 4G / 4G genotype of PAI-1, is a potentially reversible risk factor for pregnancy complications. The 4G / 4G genotype is prevalent in approximately 25% of the Chinese population and is strongly independently associated with adverse pregnancy outcomes. Compared with women with the 5G / 5G genotype, those with the 4G / 5G genotype have a 3- to 4-fold higher risk of recurrent miscarriage, while those with the 4G / 4G genotype have a 44- to 55-fold higher risk.

[0005] When using the existing peak height number correspondence judgment method to detect PAI-1 gene polymorphism, the existing quantity corresponding "quantitative" method is easily affected by many factors such as the difference in sequencer units, the quality of sequencing consumables, and experimental operations. The height of the sequencing peak will be ambiguous, resulting in the inability to classify the interpretation results. Therefore, it is urgent to design a reading method for pyrophosphate sequencing results that can accurately interpret PAI-1 gene polymorphism. Summary of the Invention

[0006] In view of the above problems existing in the prior art, the purpose of the present invention is to obtain a method for interpreting SNP gene detection based on pyrophosphate sequencing that can "qualitatively" interpret the results.

[0007] To achieve one of the above-mentioned objectives, the present invention adopts the following technical solution for the interpretation method of SNP gene detection based on pyrosequencing:

[0008] The interpretation method is used to interpret the pyrophosphate sequencing results of the rs1799762 site of the PAI-1 gene, and during sequencing, the sequence to be tested is detected according to the "TACACG" base sequence.

[0009] Preferably, the sequences of the amplification primers used in the interpretation method are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

[0010] Preferably, the sequencing primer sequence of the interpretation method is shown in SEQ ID NO: 3 in the sequence listing.

[0011] Specifically, the interpretation method includes the following steps:

[0012] a) Designing specific amplification primers and sequencing primers based on the gene to be tested, PAI-1;

[0013] b) Sample extraction: Lysis buffer, proteinase K, and eluent are sequentially added to the whole blood sample, followed by centrifugation to obtain the precipitate, and adsorption to obtain the DNA sample to be tested;

[0014] c) PCR amplification: Add the DNA sample to be tested and the upstream and downstream primers in step b) into an amplification tube in proportion, and perform amplification in 50 μL of the reaction system;

[0015] d) Preparation of single strands: Adding a binding solution containing beads to the amplified product, purifying and washing to obtain single strands of DNA;

[0016] e) Pyrophosphate sequencing: Sequencing enzyme and sequencing substrate are added to the single-stranded DNA product prepared in step d), and dATPaS, dTTP, dGTP, and dCTP are added in sequence for pyrophosphate sequencing. The type of gene polymorphism is determined based on the peak position.

[0017] Preferably, the step b) is specifically as follows: adding lysis solution and proteinase K to the whole blood sample in sequence, placing the sample in a metal bath at 56°C for 20 minutes and then leaving it at room temperature, adding isopropanol and briefly centrifuging to collect droplets on the tube wall, adding an eluent containing isopropanol and an eluent containing anhydrous ethanol in sequence after adsorption centrifugation, centrifuging to obtain a precipitate, adding a protein precipitant after adsorption, and incubating and eluting to obtain a DNA sample to be tested.

[0018] Step c) The PCR amplification system specifically includes: 10×PCR buffer: 5 μL; d(U)NTPs: 4 μL; PAI-1-4G / 5G-F: 1 μL; PAI-1-4G / 5G-R: 1 μL; Taq enzyme: 0.4 μL; UDG: 1.6 μL; ddH2O: 35 μL; sample DNA: 2 μL.

[0019] Step c) PCR amplification method is as follows: start at 25°C for 10 min, heat at 95°C for 5 min, denaturation at 95°C for 25 s, annealing at 60°C for 25 s, extension at 72°C for 25 s for a total of 50 cycles, and termination at 72°C for 5 min.

[0020] The step d) specifically comprises: adding a binding solution containing microbeads to the amplified product, shaking and mixing, and then transferring to a purification column. After centrifugation, adding a wash buffer to the purification column, repeating the washing and centrifugation three times, inserting the washed purification column into a new tube containing sequencing primers and annealing buffer, adding a denaturing solution and letting it stand, centrifuging and discarding the purification column, and collecting the single-stranded product.

[0021] Step e) specifically comprises: transferring the single-stranded product to a sequencing tube, adding 2.5 μL of sequencing enzyme and 2.5 μL of sequencing substrate to each sequencing tube, taking a dNTP array tube, adding 20 μL of dATPaS, 20 μL of dTTP, 20 μL of dGTP, and 20 μL of dCTP in sequence from the smooth end to the blunt end, gently tapping the bottom of the array tube against the table to spread the bases evenly on the bottom of the array tube, and then sequencing.

[0022] During sequencing, pyrophosphate sequencing detects the sequence to be tested according to the "TACACG" base sequence. From the first A peak from left to right in the peak graph, two consecutive peaks are judged as 4G / 4G type, and three consecutive peaks are judged as 4G / 5G type. From the first C peak from left to right in the peak graph, two consecutive peaks are judged as 5G / 5G type.

[0023] The existing interpretation method of SNP gene detection based on pyrosequencing is similar to the "quantitative" method. This "quantitative" method is easily affected by many factors such as differences in sequencer units, the quality of sequencing consumables, and experimental operations. Most cases fall within the ranges of 15%-35% and 65%-85%, and may even cause misjudgment between different genotypes, such as misjudging 4G / 4G type as 4G / 5G type, and misjudging 4G / 5G type as 5G / 5G type, etc., increasing the workload of re-inspection.

[0024] Compared with existing sequencing and reading methods, the reading method adopted in the present invention can intuitively judge the genetic polymorphism of PAI-1 by peak shape, reducing the misjudgment between the three genotypes. The "qualitative" judgment results are intuitive and accurate, greatly reducing the misjudgment rate of pyrophosphate sequencing results, increasing the scope of application, reducing labor costs, and also providing ideas for discovering more similar "qualitative" judgment gene sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a theoretical peak diagram of the detection results of the three sites of the interpretation method provided by the present invention;

[0026] Figure 2 This is the actual detection diagram of the three sites of the interpretation method provided by the present invention;

[0027] Figure 3 It is the theoretical peak diagram of the detection results of the three sites of the existing interpretation method;

[0028] Figure 4 It is the actual detection diagram of the existing interpretation method. DETAILED DESCRIPTION

[0029] The following examples further illustrate the SNP gene detection and interpretation method based on pyrosequencing provided by the present invention in detail. The following examples are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0031] The gene to be tested is PAI-1, polymorphic site: rs1799762, GGGG / GGGGG (abbreviated: 4G / 5G), GGGG=0.52, GGGGG=0.48. The specific sequence is shown below:

[0032]

[0033] The flanking sequences of the polymorphic sites are shown in bold in the text:

[0034]

[0035] Amplification primers:

[0036] PAI-1-4G / 5G-F---TGACACAAGAGAGCCCTCAGG (as shown in SEQ ID NO: 1)

[0037] PAI-1-4G / 5G-R--TTTCCCTCATCCCTGCCAT (as shown in SEQ ID NO: 2)

[0038] Sequencing primer sequences:

[0039] 5'-TGATACACGGCTGACTCCCC-3' (as shown in SEQ ID NO: 3 in the sequence listing)

[0040] 1. Required instruments and equipment

[0041] name model name model Mini Mixer MIX-30S 96-well plate mixer MX-M Handheld centrifuge D1008 Pyrosequencer PYROSEQ-E16 High-speed centrifuge D3024 / H1-16K PCR instrument PR-96E Constant temperature metal bath HB120-S

[0042] 2. Reagents and consumables

[0043] Nucleic acid extraction or purification reagents (registration number: Hunan Changxiebei 20190410), sequencing reaction universal kit (registration number: Hunan Changxiebei 20190409), microbeads (high-efficiency agarose reagent)

[0044] 3. Nucleic Acid Extraction

[0045] 1) Take 200ul of whole blood and transfer it to a 1.5ml centrifuge tube;

[0046] 2) Add 300 μl of lysis buffer and 10 μl of proteinase K (20 mg / ml) to the centrifuge tube, shake to mix, centrifuge briefly to collect droplets on the tube wall, and place in a metal bath at 56°C for 20 min;

[0047] 3) Remove the centrifuge tube from the metal bath, let it stand at room temperature, add 320 μl of isopropanol, mix well, and centrifuge briefly to collect droplets on the tube wall;

[0048] 4) Transfer 700 μl of the liquid from step 3) to the adsorption column, centrifuge at 8000 rpm for 30 seconds, and discard the waste liquid;

[0049] 5) If the transfer cannot be completed in one go, repeat step 4 until all the liquid in the centrifuge tube is transferred.

[0050] 6) Add 500 μl of isopropanol-containing eluent to the adsorption column, centrifuge at 8000 rpm for 30 seconds, and discard the waste liquid;

[0051] 7) Add 500 μl of elution solution containing anhydrous ethanol to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid;

[0052] 8) Repeat step 7);

[0053] 9) Centrifuge the empty adsorption column at 12000 rpm for 1 min and let it dry for 3-5 min;

[0054] 10) Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 60 μl of protein precipitation reagent, incubate for 5 minutes, and centrifuge at 12,000 rpm for 1 minute;

[0055] 11) Elute with eluent, discard the adsorption column, and store the DNA at -20℃.

[0056] RNase A was not used in this example. The extracted DNA may contain RNA. If RNA is not needed, RNase A can be used to remove it.

[0057] According to the instructions, the DNA extraction purity should be between OD 1.6 and 2.0; the genomic DNA yield from 200ul whole blood sample should not be less than 1ug.

[0058] 4. PCR Amplification

[0059] 1) PCR system (50 μL):

[0060] 10× PCR buffer: 5 μL;

[0061] d(U)NTPs (2.5mM each): 4μL;

[0062] PAI-1-4G / 5G-F (10μM): 1μL;

[0063] PAI-1-4G / 5G-R (10μM): 1μL;

[0064] Taq enzyme (5U / μL): 0.4 μL;

[0065] UDG (1U / μL): 1.6μL;

[0066] ddH2O: 35 μL;

[0067] Sample DNA: 2 μL.

[0068] 2) Transfer the prepared reaction system to the PCR instrument and perform the amplification reaction according to the following procedure:

[0069]

[0070] After the reaction is completed, centrifuge instantly and perform agarose gel electrophoresis or single-strand preparation.

[0071] 5. Preparation of single strands

[0072] 1) Add 40 μL of binding buffer (containing beads) to the PCR tube containing the PCR product, place on a shaker, and shake at 1100 rpm for 15 minutes.

[0073] 2) Place the purification column on the collection tube. After shaking, pipette the mixture three times to mix thoroughly. Transfer the mixture to the purification column and centrifuge at 7000 × g for 1 min.

[0074] 3) Add 150 μL of wash buffer to the purification column and centrifuge at 7000 × g for 1 min (this step is repeated 3 times in total).

[0075] 4) Take new clean EP tubes, add 6 μL of annealing buffer to each EP tube, and then add 1 μL of sequencing primer.

[0076] 5) After washing three times, remove the purification column and insert it into the EP tube containing sequencing primers. Add 22uL of denaturing solution to each purification column, let it stand for 5 minutes, and centrifuge at 7000×g for 1 minute. Collect the single-stranded product in the EP tube and discard the purification column.

[0077] 6. Sequencing

[0078] 1) Transfer the single-stranded product in the EP tube to a sequencing tube and add 2.5 μL of sequencing enzyme and 2.5 μL of sequencing substrate to each sequencing tube.

[0079] 2) Take a dNTP tube and add 20 μL dATPaS, 20 μL dTTP, 20 μL dGTP, and 20 μL dCTP, starting from the rounded end and working toward the blunt end. Gently tap the bottom of the tube against the table to spread the bases evenly across the bottom.

[0080] 3) Prepare the cleaning sink: Add pure water to the cleaning sink until the brush is covered, and place absorbent paper in the designated position of the cleaning sink.

[0081] 4) Start the computer, run the iLight software, and click the "Tray In / Out" button. Once the tray is out of the way, place the wash tank, dNTP tubes, and sequencing tubes in their designated locations. Click the "Tray In / Out" button again to retract the tray.

[0082] 5) Click the "New" button. Select the project to be sequenced from the AI ​​drop-down menu and enter the corresponding number of samples. Click the "Start Sequencing" button. After carefully checking the prompt dialog box to ensure that the preparations are complete, click the "OK" button to start sequencing.

[0083] 6) When sequencing is complete, the software will pop up the "End" dialog box. Click the "OK" button. Click the "Save" button to save the sequencing results file. Click the "Output Report" button and enter the sample code and other information to save the genotype results (in Excel format).

[0084] 7) After the experiment: Click "Tray In / Out" to remove the sequencing tube, base strip tube, and water tank in sequence; click "Tray In / Out" again. After the tray is in, click "Exit" and turn off the instrument.

[0085] Example 1

[0086] According to the "TACACG" base sequence, the sequence to be tested is detected. The theoretical peak shapes of the three genotypes are as follows Figure 1 As shown, this method only relies on the area shown in the dotted box, that is, the position and number of peaks, to perform typing: the peaks at the first two bases are the 4G / 4G wild type, the peaks at all three bases are the 4G / 5G heterozygous type, and the peaks at the last two bases are the 5G / 5G mutant type.

[0087] Comparative Example 1

[0088] The above PAI-1 polymorphic sites are detected according to the "TCACG" base sequence. The theoretical peak shapes of the three genotypes are as follows: Figure 3As shown in the figure, this method relies on the ratio of the height of the C peak to the height of the A peak shown in the dotted box to perform typing. In actual application, based on a certain number of samples with known genotypes, three genotype interpretation intervals are statistically calculated, that is, C / A between 0%-15% is judged as 4G / 4G type, between 35%-65% is judged as 4G / 5G type, and between 85%-100% is judged as 5G / 5G type. When the sequencing peak decays randomly and irregularly, the positive reference peak as the denominator becomes a very tricky variable. In actual detection, it is easy to have the following Figure 4 The ambiguous situation shown makes it impossible to classify the interpretation results.

[0089] Compared with Comparative Example 1, Example 1 adopts a "qualitative" method to read the test results, and the accuracy is greatly improved, and there is basically no misreading (that is, if the 4G / 5G type is to be misjudged as the 4G / 4G type, the first A peak needs to be raised to the highest and equal to the C peak, and the third A peak needs to be reduced to the lowest to the baseline. One rise and one fall are two completely different trends, which are theoretically impossible.) The actual detection diagram of the three genotypes of PAI-1 gene polymorphism (as shown in Figure 2 The feasibility of this reading method is also demonstrated.

[0090] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for interpreting SNP gene detection based on pyrosequencing, characterized in that: The interpretation method is used to interpret the pyrophosphate sequencing results of the rs1799762 site of the PAI-1 gene, and the sequence to be tested is detected according to the "TACACG" base sequence during sequencing.

2. The method for interpreting SNP gene detection based on pyrosequencing according to claim 1, characterized in that: The interpretation method comprises the following steps: a) Designing specific amplification primers and sequencing primers based on the gene to be tested, PAI-1; b) Sample extraction: add lysis buffer, proteinase K, and eluent to the whole blood sample in sequence, and centrifuge to obtain the precipitate. Adsorption to obtain the DNA sample to be tested; c) PCR amplification: Add the DNA sample to be tested and the upstream and downstream primers in step b) into an amplification tube in proportion, and perform amplification in 50 μL of the reaction system; d) Preparation of single strands: Adding a binding solution containing beads to the amplified product, purifying and washing to obtain single strands of DNA; e) Pyrophosphate sequencing: Sequencing enzyme and sequencing substrate are added to the single-stranded DNA product prepared in step d), and dATPaS, dTTP, dGTP, and dCTP are added in sequence for pyrophosphate sequencing. The type of gene polymorphism is determined based on the peak position.

3. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: The sequences of the amplification primers used in the interpretation method are shown in the sequence listing as SEQ ID NO: 1 and SEQ ID NO:

2.

4. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: The sequencing primer sequence of the interpretation method is shown in the sequence listing as SEQ ID NO:

3.

5. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: The step b) specifically comprises: sequentially adding a lysis solution and proteinase K to the whole blood sample, adding isopropanol after metal bath for adsorption and centrifugation, sequentially adding an eluent containing isopropanol and an eluent containing anhydrous ethanol, centrifuging to obtain a precipitate, adding a protein precipitant after adsorption, and incubating and eluting to obtain a DNA sample to be tested.

6. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: Step c) The PCR amplification system specifically includes: 10×PCR buffer: 5 μL; d(U)NTPs: 4 μL; PAI-1-4G / 5G-F: 1 μL; PAI-1-4G / 5G-R: 1 μL; Taq enzyme: 0.4 μL; UDG: 1.6 μL; ddH2O: 35 μL; sample DNA: 2 μL.

7. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: Step c) PCR amplification method is as follows: start at 25°C for 10 min, heat at 95°C for 5 min, denaturation at 95°C for 25 s, annealing at 60°C for 25 s, extension at 72°C for 25 s for a total of 50 cycles, and termination at 72°C for 5 min.

8. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: The step d) specifically comprises: adding a binding solution containing microbeads to the amplified product, shaking and mixing, and then transferring to a purification column. After centrifugation, adding a wash buffer to the purification column, repeating the washing and centrifugation, inserting the washed purification column into a new tube containing sequencing primers and annealing buffer, adding a denaturing solution, and then standing. Centrifuging and discarding the purification column to collect the single-stranded product.

9. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: The step e) specifically comprises: transferring the single-stranded product to a sequencing tube, adding 2.5 μL of sequencing enzyme and 2.5 μL of sequencing substrate to each sequencing tube, taking a dNTP array tube, adding 20 μL of dATPaS, 20 μL of dTTP, 20 μL of dGTP, and 20 μL of dCTP in sequence from the smooth end to the blunt end, gently tapping the bottom of the array tube against the table to spread the bases evenly on the bottom of the array tube, and then sequencing.

10. The method for interpreting SNP gene detection based on pyrosequencing according to claim 2, characterized in that: During sequencing, pyrophosphate sequencing detects the sequence to be tested according to the "TACACG" base sequence. Starting from the first A peak from left to right in the peak pattern graph, two consecutive peaks are determined to be 4G / 4G type, and three consecutive peaks are determined to be 4G / 5G type. Starting from the first C peak from left to right in the peak pattern graph, two consecutive peaks are determined to be 5G / 5G type.

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