POLE gene mutation detection kit

Abstract

The invention relates to a POLE gene mutation detection kit. The kit comprises a reaction solution A, a reaction solution B1, a reaction solution B2, a reaction solution B3, a reaction solution B4, a sequencing primer B1, a sequencing primer B2, a sequencing primer B3 and a sequencing primer B4, the kit is used for detecting the mutation condition of the human POLE gene. According to the present invention, the unique primer probe and the unique amplification system are provided, the 21 mutation types of the POLE gene exonuclease structural domain can be specifically and highly sensitively detected only through the 5 ng DNA, and the detection sensitivity is as low as 1%; compared with the defects that the traditional Sanger sequencing detection sensitivity is low, the number of detection sites of a PCR method is small, NGS operation is complex, and the price is high, the method has obvious advantages in clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a POLE gene mutation detection kit. Background Art

[0002] A gene is a DNA sequence that carries genetic information and is the fundamental unit of inheritance that controls traits. Genetic variation is a key factor in human disease. Genetic testing can assess a patient's health status and disease risk, and also serves as an important basis for clinical diagnosis and treatment.

[0003] The POLE gene is located at human 12q24.3, with a cDNA length of 63,604 bp. It is the largest catalytic subunit of DNA polymerase ε. It possesses two main catalytic activities: a DNA-based polymerase activity and an exonuclease proofreading activity, both of which play a crucial role in cellular DNA replication and base mismatch repair. Germline or somatic mutations in the gene encoding the POLE exonuclease region can lead to a loss of POLE exonuclease proofreading activity, increasing genomic instability and preventing the recognition and excision of mismatches. This leads to an abnormally high number of genomic mutations, cellular damage, and an increased risk of cancer. Recent studies have revealed that mutations in the POLE exonuclease region have been detected in a variety of tumors, including endometrial cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, and high-grade gliomas. Endometrial cancer is the malignant tumor with the highest prevalence of POLE mutations, at 7% to 12%.

[0004] With the deepening of research on the POLE gene, POLE gene mutation detection is being used more and more widely in clinical practice, such as molecular typing of endometrial cancer, and medication guidance for solid tumors such as colorectal cancer and small intestinal cancer.

[0005] Currently, the main genetic testing technologies used in clinical practice include PCR, first-generation sequencing, and second-generation sequencing. PCR can only detect a few variants at a time, making it prone to missed detections; standard first-generation sequencing has low sensitivity and is prone to false negatives; and NGS is complex, time-consuming, and expensive, making it difficult to implement. Therefore, there is an urgent need for a new method and kit to simultaneously detect multiple pathogenic sites within the exonuclease domain of the POLE gene, while also striking a balance between sensitivity, cycle time, cost, and affordability. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a POLE gene mutation detection kit, which can simultaneously perform highly sensitive detection of 21 mutations of the human POLE gene with only 5ng of DNA.

[0007] The present invention provides a POLE gene mutation detection kit, which includes reaction solution A, reaction solution B1, reaction solution B2, reaction solution B3, reaction solution B4, and sequencing primers B1, sequencing primer B2, sequencing primer B3, and sequencing primer B4. The kit is used to detect mutations in the human POLE gene.

[0008] The specific variations are shown in the following table: Preferably, the reaction solution A comprises PCR master mix, thermosensitive UNG / UDG enzyme, dUTP, formamide, and primers SEQ NO. 1-8.

[0009] Preferably, the reaction solution B1 contains PCR master mix, dUTP, and primer probes SEQ NO.9-11; wherein the probe SEQ NO.11 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

[0010] Preferably, the reaction solution B2 contains PCR master mix, dUTP, and primer probes SEQ NO.12-14; wherein the probe SEQ NO.14 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

[0011] Preferably, the reaction solution B3 contains PCR master mix, dUTP, and primer probes SEQ NO.15-17; wherein the probe SEQ NO.17 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

[0012] Preferably, the reaction solution B4 contains PCR master mix, dUTP, and primer probes SEQ NO.18-20; wherein the probe SEQ NO.20 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

[0013] Preferably, the sequence of the sequencing primer B1 is SEQ NO. 21, and the concentration is 2-10 μM.

[0014] Preferably, the sequence of the sequencing primer B2 is SEQ NO. 22, and the concentration is 2-10 μM.

[0015] Preferably, the sequence of the sequencing primer B3 is SEQ NO. 23, and the concentration is 2-10 μM.

[0016] Preferably, the sequence of the sequencing primer B4 is SEQ NO. 24, and the concentration is 2-10 μM.

[0017] Preferably, the kit is used for two-step amplification, and the specific conditions are as follows: The first step of amplification: Second step amplification: The principle of the present invention is as follows: first, the four exon regions of EXON9, ENOX11, EXON13, and EXON14 of the POLE gene are enriched through a first round of primer amplification, and then four sets of internal primers and wild-type inhibition probes are designed for the target sites in the four exon regions, and the probes must cover the target detection site. The 3' end of the primer on the target site side has 2-10bp overlapping with the 5' end of the probe, and the probe TM value is higher than that of the primer. The probe is 3'-end blocked by one or a combination of 3' end base mismatch, phosphorylation, ddC modification, steric modification, MGB modification, etc., so that it will not extend after binding to the template. When the PCR reaction is performed, the probe preferentially binds to the wild-type site region, resulting in low binding efficiency of the same-direction amplification primer or inability to extend after binding due to steric hindrance, thereby inhibiting the amplification of the wild-type sample and increasing the proportion of mutants in the sample. The amplified sample is then tested by a first-generation sequencing method, which can significantly improve the sensitivity of mutation detection.

[0018] Beneficial effects (1) The present invention can simultaneously detect 21 mutations of the POLE gene with high detection throughput; (2) The present invention only requires 5ng of DNA for effective detection, which can solve the problem of difficulty in detecting trace samples such as puncture; (3) Conventional first-generation sequencing technology can only detect mutations with a mutation frequency of more than 20%, while the present invention can detect mutations as low as 1%, with high detection sensitivity; (4) The present invention adopts the UNG / UDG anti-pollution system, which can effectively avoid sample cross-contamination caused by aerosols, etc., and further ensure the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-5 This is the sequencing result of the target site in the EXON09 region of the POLE gene.

[0020] Figure 6-10 This is the sequencing result of the target site in the EXON11 region of the POLE gene.

[0021] Figure 11-15 This is the sequencing result of the target site in the EXON13 region of the POLE gene.

[0022] Figure 16-21 This is the sequencing result of the target site in the EXON14 region of the POLE gene. DETAILED DESCRIPTION

[0023] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0024] Example 1 1. Kit Preparation: Reaction solution A formula: PCR master mix 10μL, thermosensitive UNG / UDG enzyme 0.5μL, dUTP (100mM) 0.05μL, formamide 1.0μL, primers SEQ NO.01-08 (100μM) 0.05μL each, add water to 15μL.

[0025] Reaction solution B1 formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.09-10 (100μM) 0.05μL each, probe SEQ NO.11 (100μM) 0.5μL, add water to 9μL.

[0026] Reaction solution B2 formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.12-13 (100μM) 0.05μL each, probe SEQ NO.14 (100μM) 0.5μL, add water to 9μL.

[0027] Reaction solution B3 formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.15-16 (100μM) 0.05μL each, probe SEQ NO.17 (100μM) 0.5μL, add water to 9μL.

[0028] Reaction solution B4 formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.18-19 (100μM) 0.05μL each, probe SEQ NO.20 (100μM) 0.5μL, add water to 9μL.

[0029] Sequencing primer B1 formula: Primer SEQ NO.21 (100 μM), diluted to 5 μM with ultrapure water.

[0030] Sequencing primer B2 formula: Primer SEQ NO. 22 (100 μM), diluted to 5 μM with ultrapure water.

[0031] Sequencing primer B3 formula: Primer SEQ NO. 23 (100 μM), diluted to 5 μM with ultrapure water.

[0032] Sequencing primer B4 formula: Primer SEQ NO. 24 (100 μM), diluted to 5 μM with ultrapure water.

[0033] The primer probe sequences are as follows: The probe is blocked at the 3' end by modifications such as base mismatch, phosphorylation, ddC, MGB, spacer, etc. The modification can be any one or a combination of multiple modifications.

[0034] SEQ NO.1-8 is used to amplify and enrich the target region of the POLE gene; SEQ NO.9, SEQ NO.10, and SEQ NO.11 are used in combination for amplification of POLE gene EXON09, and SEQ NO.21 is the sequencing primer for POLE gene EXON09; SEQ NO.12, SEQ NO.13, and SEQ NO.14 are used in combination for amplification of POLE gene EXON11, and SEQ NO.22 is a sequencing primer for POLE gene EXON11; SEQ NO.15, SEQ NO.16, and SEQ NO.17 are used in combination for amplification of POLE gene EXON13, and SEQ NO.23 is a sequencing primer for POLE gene EXON13; SEQ NO.18, SEQ NO.19, and SEQ NO.20 are combined for amplification of POLE gene EXON14, and SEQ NO.24 is a sequencing primer for POLE gene EXON14.

[0035] 2. Inspection methods: 1. Sample Preparation The following samples were prepared using human wild-type genomic DNA and mutant genomic DNA for detection and verification.

[0036] Amplification step 1: Take 15 μL of Reaction Solution A for each sample, add 5 ng of the corresponding DNA, and make up to 20 μL with water. Vortex to mix, centrifuge, and then place in a PCR instrument for reaction. The reaction conditions are as follows: After the first step of amplification is completed, the amplified product is diluted 20 times with ultrapure water for later use.

[0037] Second step amplification: Take 9 μL each of reaction solution B1, reaction solution B2, reaction solution B3, and reaction solution B4, add 1 μL of the diluted first step amplification product to each of them, vortex to mix, centrifuge, and react on a PCR instrument. The reaction conditions are as follows: After the second amplification step, the amplified products were purified and sequenced. The corresponding sequencing primers for the amplified products of reaction solutions B1, B2, B3, and B4 were sequencing primer B1, B2, B3, and B4, respectively. After the sequencing reaction, the purified products were purified and sequenced using a first-generation sequencer.

[0038]

[0039] Conclusion: The present invention can detect the above-mentioned mutation sites of the human POLE gene with high sensitivity and accuracy.

Claims

1. A POLE gene mutation detection kit, characterized by: The kit includes reaction solution A, reaction solution B1, reaction solution B2, reaction solution B3, reaction solution B4 and sequencing primers B1, sequencing primer B2, sequencing primer B3, and sequencing primer B4; the kit is used to detect mutations in the human POLE gene.

2. The POLE gene mutation detection kit according to claim 1, characterized in that: The reaction solution A contains PCR master mix, thermosensitive UNG / UDG enzyme, dUTP, formamide, and primers SEQ NO. 1-8.

3. The POLE gene mutation detection kit according to claim 1, characterized in that: The reaction solution B1 contains PCR master mix, dUTP, and primer probes SEQ NO.9-11; wherein the probe SEQ NO.11 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

4. The POLE gene mutation detection kit according to claim 1, characterized in that: The reaction solution B2 contains PCR master mix, dUTP, and primer probes SEQ NO.12-14; wherein the probe SEQ NO.14 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

5. The POLE gene mutation detection kit according to claim 1, characterized in that: The reaction solution B3 contains PCR master mix, dUTP, and primer probes SEQ NO.15-17; wherein the probe SEQ NO.17 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

6. The POLE gene mutation detection kit according to claim 1, characterized in that: The reaction solution B4 contains PCR master mix, dUTP, and primer probes SEQ NO.18-20; wherein the probe SEQ NO.28 is 3'-end blocked by one or more combinations of 3'-end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, so that it will not extend after binding to the template.

7. The POLE gene mutation detection kit according to claim 1, characterized in that: The sequence of the sequencing primer B1 is SEQ NO. 21, and the concentration is 2-10 μM.

8. The POLE gene mutation detection kit according to claim 1, characterized in that: The sequence of the sequencing primer B2 is SEQ NO. 22, and the concentration is 2-10 μM.

9. The POLE gene mutation detection kit according to claim 1, characterized in that: The sequence of the sequencing primer B3 is SEQ NO. 23, and the concentration is 2-10 μM.

10. The POLE gene mutation detection kit according to claim 1, characterized in that: The sequence of the sequencing primer B4 is SEQ NO. 24, and the concentration is 2-10 μM.

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