Primer for detecting Exon10 hotspot mutation of MPL gene and application

Abstract

The invention provides a primer for detecting Exon10 hot spot mutation of an MPL gene and application of the primer, the primer for detecting the Exon10 hot spot mutation of the MPL gene comprises amplification primers and sequencing primers, the sequences of the amplification primers are 5 '-AGTAGGGGCTGGCTGGATGA-3' and 5 '-ACCTGACGCAGATTGGATGG-3', and the sequences of the sequencing primers are 5 '-AGTAGGGGCTGGGATGG-3', 5 '- And the sequences of the sequencing primers are 5 '-GGCCGAAGTCTGACCCTT-3' and 5 '-GCGGTATAGTGGGCGTGTT-3'. The PCR amplification primers and the sequencing primers are separated, one pair of amplification primers is firstly used for PCR amplification, and the other pair of sequencing primers is used for sequencing, so that a single peak with a better sequencing effect is obtained, the mutation condition is accurately judged, and the sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a primer for detecting hotspot mutations in Exon 10 of an MPL gene and an application thereof. Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a group of neoplastic diseases that arise from hematopoietic stem cells, undergo relatively mature differentiation, and are characterized by clonal hyperproliferation of one or more myeloid hematopoietic lineages. The pathogenic mechanism of classic MPNs is primarily due to mutations in genes involved in the JAK / STAT pathway, driving excessive myeloid cell proliferation. MPL is one such gene. The MPL gene (myeloproliferative leukemia gene) encodes the thrombopoietin receptor, which promotes cell growth, proliferation, and differentiation. It is part of the TPO-JAK2-STAT signaling pathway and participates in the JAK2-STAT signaling pathway. Mutations in the MPL gene can aberrantly activate downstream signaling factors, contributing to tumor development and progression. Common MPL gene mutations include W515L and W515K, W515A, W515S, W515R, and S505N, all clustered in Exon 10. These mutations are observed in 3-5% of ET and 8-10% of PMF patients.

[0003] Currently, the primary method used to detect mutations in Exon 10 of the MPL gene is Sanger sequencing. This method is extremely common in clinical practice and is considered the gold standard for detecting gene mutations. Due to the high GC content of Exon 10 of the MPL gene, primer design is relatively difficult, resulting in suboptimal sequencing results, including the presence of spurious bands and sequencing failures. Summary of the Invention

[0004] In view of this, the present invention provides a primer and application for detecting hotspot mutations in Exon10 of the MPL gene. The PCR amplification primers and sequencing primers are separated. A pair of amplification primers are first used for PCR amplification, and another pair of sequencing primers are used for sequencing to obtain a single peak with better sequencing effect.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a primer for detecting hotspot mutations in Exon 10 of the MPL gene, comprising an amplification primer and a sequencing primer, wherein the amplification primer is: Amplification primer MPL-10-F: 5′-AGTAGGGGCTGGCTGGATGA-3′, primer length 20 bp, Tm value 62.55, GC% 60%; Amplification primer MPL-10-R: 5'-ACCTGACGGCAGATTGGATGG-3', primer length is 21 bp, Tm value is 62.46, GC% is 57.14%; The sequencing primers include: Sequencing primer MPL-10-F: 5′-GGCCGAAGTCTGACCCTTT-3′ Sequencing primer MPL-10-R: 5′-GCGGTATAGTGGGCGTGTT-3′.

[0006] In a second aspect, the present invention provides a kit for detecting hotspot mutations in Exon 10 of the MPL gene, the kit comprising the above-mentioned primers.

[0007] Furthermore, the kit also includes an enzyme digestion system, which includes the following components: sterilized deionized water, calf intestinal alkaline phosphatase (CIP), and exonuclease I (E. coli).

[0008] In some specific embodiments, preferably, the volume ratio of sterile deionized water, calf intestinal alkaline phosphatase, and nuclease exonuclease I in the kit is 2.1:0.1:0.8.

[0009] In a third aspect, the present invention provides a method for detecting hotspot mutations in Exon 10 of the MPL gene using the above-mentioned kit, comprising the following steps: S1. Extract DNA from the sample and perform PCR amplification; S2. Perform electrophoresis on the PCR amplification product. If a single band of 500 bp (PCR amplification product length 469 bp, Tm value: 94.7, GC%: 64.8%) is detected, subsequent sequencing can be performed. S3. The products to be sequenced are purified by enzyme digestion and further amplified by sequencing PCR. After purification again, they are sequenced and analyzed using a gene sequencer to obtain the mutation status.

[0010] In some specific embodiments, preferably, the PCR program in the sequencing PCR amplification is as follows: 96°C / 60S; 96°C / 10S, 50°C / 5S, a total of 25 cycles; 60°C / 240S.

[0011] Fourthly, the application of the above primers and kits in the preparation of MPL gene Exon10 hotspot mutation detection products.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention can detect all mutations in Exon 10 of the MPL gene, including the most common W515 mutation, as well as other sites such as the S505N mutation and the co-mutation of S505N and G509V. Furthermore, this technology separates PCR amplification primers from sequencing primers, using one pair of amplification primers for PCR amplification and another pair of sequencing primers for sequencing. This yields a single peak with excellent sequencing results, enabling accurate mutation determination with high detection sensitivity, providing a diagnostic basis for subsequent treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the gel electrophoresis diagram of the amplified product of the sample in Example 1 of the present invention.

[0014] Figure 2 This is a peak diagram of the sequencing results obtained by direct sequencing using amplification primer F in Example 1 of the present invention.

[0015] Figure 3 This is a peak diagram of the sequencing results obtained by direct sequencing using amplification primer R in Example 1 of the present invention.

[0016] Figure 4 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer F in Example 1 of the present invention (negative, no mutation).

[0017] Figure 5 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (negative, no mutation).

[0018] Figure 6 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer F in Example 1 of the present invention (positive, W515L mutation).

[0019] Figure 7 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, W515L mutation).

[0020] Figure 8 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer F in Example 1 of the present invention (positive, W515A mutation).

[0021] Figure 9 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, W515A mutation).

[0022] Figure 10 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, W515K mutation).

[0023] Figure 11 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, S505N mutation).

[0024] Figure 12 This is the peak graph of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, W515R mutation).

[0025] Figure 13 This is a peak diagram of the sequencing results obtained by sequencing with sequencing primer F in Example 1 of the present invention (positive, S505N and G509V co-mutation).

[0026] Figure 14 This is a peak diagram of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, S505N and G509V co-mutation).

[0027] Figure 15 This is the peak diagram of the sequencing results obtained by sequencing with sequencing primer R in Example 1 of the present invention (positive, W515L low-frequency mutation). DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0029] Example 1 This example provides a primer for detecting hotspot mutations in Exon 10 of the MPL gene and a specific experimental process, as follows: Currently, the primary method used to detect mutations in Exon 10 of the MPL gene is Sanger sequencing. This method is extremely common in clinical practice and is considered the gold standard for detecting gene mutations. Due to the high GC content of Exon 10 of the MPL gene, primer design is relatively difficult, resulting in suboptimal sequencing results, including the presence of spurious bands and sequencing failures.

[0030] Design of amplification primers: PCR amplification primers were designed using Primer5 software. Based on parameters such as primer length, Tm value, and GC content, the following amplification primers were designed: Amplification primer MPL-10-F: 5'-AGTAGGGGCTGGCTGGATGA-3', primer length: 20 bp, Tm value: 62.55, GC%: 60%; Amplification primer MPL-10-R: 5'-ACCTGACGGCAGATTGGATGG-3', primer length: 21 bp, Tm value: 62.46, GC%: 57.14%; The PCR amplification product was 469 bp in length, with a Tm value of 94.7 and a GC% of 64.8%. After BLAST analysis, there were no interfering bands of similar size for this pair of primers, and the primers were usable.

[0031] Sequencing primers: A pair of sequencing primers was redesigned within the PCR amplification product, also using Primer 5 software. The forward and reverse primers were selected to ensure that they could fully detect MPL-Exon 10 and be within the PCR amplification product. The following sequencing primer pair was found: Sequencing primer MPL-10-F: 5′-GGCCGAAGTCTGACCCTTT-3′; Sequencing primer MPL-10-R: 5′-GCGGTATAGTGGGCGTGTT-3′.

[0032] The specific experimental steps are as follows: 1. DNA Extraction DNA was extracted from bone marrow / peripheral blood using the Biomed blood genomic DNA extraction kit and used for PCR amplification.

[0033] 2. PCR Amplification 2.1 Primer preparation: According to the manufacturer's OD of the synthesized primers 260 If the values ​​are different, prepare the primers into a stock solution with a final concentration of 100 μM / L, and then prepare the working solution with a primer concentration of 5 pmoL / μL.

[0034] 2.2 PCR system and PCR reaction procedure are shown in Tables 1 and 2.

[0035] Table 1 PCR system details

[0036] Table 2 Details of PCR reaction conditions

[0037] 2.3 PCR product electrophoresis detection: The sample band is about 500bp and the band is single (see Figure 1 ), the amplified product can be sent to the detection sequence.

[0038] 3. Sequencing 3.1. Purification: PCR products were purified using enzyme digestion. The enzyme digestion system and procedure are shown in Tables 3 and 4.

[0039] Table 3 Enzyme digestion system details

[0040] Table 4 Enzyme digestion reaction conditions

[0041] 3.2. Sequencing PCR: After product purification, sequencing PCR was performed. The specific PCR system and sequencing procedure are shown in Tables 5 and 6.

[0042] Table 5 Sequencing PCR system details

[0043] Table 6 Sequencing PCR program details

[0044] 3.3. Purification of PCR products after sequencing 3.3.1 Add 1µl of 125 mM EDTA to each well and 1µl of 3M NaAc to each well. Note: If these two reagents are mixed, mix them immediately before use and thoroughly.

[0045] 3.3.2 Add approximately 25µL of 100% alcohol to each well to flush the EDTA and NaAC to the bottom of the tube. Seal the tube, vortex to mix, centrifuge briefly, and let stand at room temperature for at least 15 minutes.

[0046] 3.3.3 Centrifuge at 2000-3000g for 45 minutes. Invert the 96-well plate and centrifuge until the plate reaches approximately 185g (the total time from starting the centrifuge to reaching approximately 185g is approximately 1 minute). Note: When centrifuging inverted, place a clean piece of paper under the plate. The paper must be high-quality, free of powder and fluorescent substances.

[0047] 3.3.4 Add approximately 35 µL of 70% ethanol to each tube and centrifuge at 2000-3000 g for 20 min. Invert the 96-well plate and centrifuge until the centrifuge reaches approximately 185 g (the total time from starting the centrifuge to reaching approximately 185 g is approximately 1 min).

[0048] 3.3.5 Dry in a vacuum desiccator at 45°C for 15 minutes. Add 10µL of Hi-Di Formamide to dissolve the DNA (mix the Hi-Di well before use). After adding the Hi-Di, remix the sample and briefly centrifuge. If testing cannot be performed immediately, seal and refrigerate. Note: Observe that the alcohol has been completely removed. If not, extend the drying time to ensure complete drying. Avoid repeated freeze-thaw cycles of the Hi-Di Formamide and avoid using it after prolonged storage. For detailed instructions, refer to the Hi-Di Formamide package insert.

[0049] 3.3.6 The dissolved samples need to be denatured at 95℃ for 4 minutes, then placed on ice and transferred to the sequencer.

[0050] 4. Sequencing with the 3500xl Genetic Sequencing Analyzer: Turn on the device, verify that it is operating normally, and check the system status. Perform the test according to the 3500xl Genetic Analyzer user manual.

[0051] 5. Results Analysis The results were analyzed using Mutation Surveyor software. Once the mutation site was found, the location of the mutation was analyzed using VECTOR NTI software.

[0052] Through the results analysis: only one pair of primers was designed and used for PCR amplification and sequencing at the same time, the peak diagram obtained is as follows Figure 2 and Figure 3 As shown, it is impossible to determine whether there is a mutation site. Further, through design, a pair of sequencing primers is added on the basis of PCR amplification primers. PCR amplification is first performed with PCR amplification primers to obtain PCR products, and then sequencing is performed with sequencing primers to obtain a single peak of ATCG bases. There is no interfering base below the peak, which can clearly determine whether there is a mutation (such as Figure 4-Figure 7 shown).

[0053] Furthermore, in order to verify the accuracy of the two pairs of primers designed in this application, five positive samples (four common W515 mutations: W515A mutation, W515K mutation, W515R mutation, W515L low-frequency mutation; two other types of mutations: S505N mutation, S505N and G509V co-mutation) were selected for testing. Specific test results are shown in Figure 8 、 9 (W515A mutation) and Figure 10 (W515K mutation), Figure 11 (S505N mutation), Figure 12 (W515R mutation), Figure 13 、 14 (S505N and G509V co-mutation), Figure 16 (W515L low-frequency mutation).

[0054] As shown in the figure, after amplifying the sample using the amplification primers provided in this application and then sequencing the product using sequencing primers, the most common W515 mutation can be detected, as well as other mutation sites (such as the S505N mutation and the co-mutation of S505N and G509V), extending the detection range. Furthermore, the low-frequency W515L mutation can also be detected, demonstrating the high sensitivity of this application. These characteristics determine that this application scheme is well suited for detecting hotspot mutations in Exon 10 of the MPL gene, providing an accurate basis for subsequent treatment.

[0055] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0056] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer for detecting hotspot mutations in Exon 10 of the MPL gene, characterized in that: The primers include amplification primers and sequencing primers, and the amplification primers are as follows: Amplification primer MPL-10-F: 5′-AGTAGGGGCTGGCTGGATGA-3′; Amplification primer MPL-10-R: 5′-ACCTGACGGCAGATTGGATGG-3′; The sequencing primers are as follows: Sequencing primer MPL-10-F: 5′-GGCCGAAGTCTGACCCTTT-3′; Sequencing primer MPL-10-R: 5′-GCGGTATAGTGGGCGTGTT-3′.

2. A kit for detecting hotspot mutations in Exon 10 of the MPL gene, characterized in that: The kit comprises the primers according to claim 1.

3. The kit according to claim 2, wherein The kit also includes an enzyme digestion system, which includes the following components: sterilized deionized water, calf intestinal alkaline phosphatase, and exonuclease I.

4. The kit according to claim 3, wherein The volume ratio of the sterilized deionized water, calf intestinal alkaline phosphatase and nuclease exonuclease I is 2.1:0.1:0.

8.

5. A method for detecting hotspot mutations in Exon 10 of the MPL gene using the kit according to any one of claims 2 to 4, comprising the following steps: S1. Extract DNA from the sample and perform PCR amplification; S2. Perform electrophoresis on the PCR amplification product. If a single band of 500 bp is detected, subsequent sequencing can be performed. S3. The products to be sequenced are purified by enzyme digestion and further amplified by sequencing PCR. After purification again, they are sequenced and analyzed using a gene sequencer to obtain the mutation status.

6. The method according to claim 5, characterized in that The PCR program in the sequencing PCR amplification was as follows: 96°C / 60S; 96°C / 10S, 50°C / 5S, for a total of 25 cycles; 60°C / 240S.

7. Use of the primers according to claim 1 and the kit according to any one of claims 2 to 4 in preparing a product for detecting hotspot mutations in Exon 10 of the MPL gene.

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