Primer pair, kit and detection method for detecting GAA repetitive sequence of FXN gene
By designing specific primer pairs and capillary electrophoresis technology, combined with fluorescent labeling and software analysis, we have achieved efficient and accurate detection of the GAA repeat sequence number of the FXN gene, solving the problems of long detection time and high cost in existing technologies, and making it suitable for clinical detection of the FXN gene.
Patent Information
- Application Number
- CN202511833700.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-04-21
AI Technical Summary
Existing technologies for detecting GAA repeat sequences in the FXN gene are limited. First-generation sequencing procedures are numerous and time-consuming, and cannot accurately determine the inconsistency in the number of GAA repeat sequences on alleles. Second-generation sequencing technology is expensive and has complex data analysis, resulting in low cost-effectiveness.
By designing specific primer pairs FXN-F and FXN-R, and combining PCR amplification and capillary electrophoresis techniques, the number of GAA repeat sequences in the FXN gene was accurately detected through fluorescent labeling and analysis using GeneMapper software.
This invention provides a simple, time-saving, and inexpensive detection method that can accurately determine the number of GAA repeat sequences in the FXN gene. It is suitable for clinical testing, solves the problems of long time and high cost of existing technologies, and can clearly identify the inconsistency in the number of GAA repeat sequences on alleles.
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Figure CN121896336A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to a primer pair, kit, and detection method for detecting GAA repetitive sequences in the FXN gene. Background Technology
[0002] Friedreich's ataxia (FA) is a common hereditary neurodegenerative disease in Europe and America. It is an autosomal recessive genetic disorder and a special type of hereditary ataxia. The disease typically begins in adolescence, but in some cases, it manifests in adulthood, and the course of the disease can last for more than 30 years. Initial symptoms include ataxia of both lower limbs, unsteady gait, and difficulty standing, often compensated by swinging the upper limbs to maintain balance. Subsequently, the neurological and muscular dysfunction spreads throughout the body, and in the late stages, death often results from heart disease. Currently, there is no effective treatment. The pathogenesis is mainly caused by homozygous mutations in the abnormal repeat amplification of the GAA trinucleotide sequence in intron 1 of the Fratxin gene (FXN) on chromosome 9. Normal individuals have 5-33 GAA repeat sequences, while premutated individuals have 34-65 GAA repeat sequences, and FA patients have 66-1200 GAA repeat sequences. In addition, a very small number are caused by compound mutations, that is, one allele of the FXN gene has an abnormal repeat amplification of the GAA trinucleotide sequence, and the other allele has a conventional point mutation (such as missense mutation, nonsense mutation, etc.).
[0003] Fratxin, encoded by the FXN gene, is an important mitochondrial protein that plays a crucial role in maintaining mitochondrial function, iron homeostasis, and the synthesis of iron-sulfur clusters. This protein is widely distributed in the spinal cord, cerebellum, and cerebral cortex of the central nervous system, as well as in non-nervous systems such as heart, skeletal muscle, liver, kidney, and pancreatic cells. GAA repeat sequences impair protein expression, leading to impaired mitochondrial iron translocation, cytoplasmic iron deficiency, and ultimately, mitochondrial iron accumulation and increased susceptibility to oxygen free radical damage. This, in turn, damages mitochondria and the respiratory chain, resulting in neurodegenerative diseases. Therefore, accurate counting of GAA repeat sequences in the FXN gene is of great significance.
[0004] Current conventional FXN gene testing is mostly based on first-generation sequencing technology. However, first-generation sequencing procedures are numerous, the testing time is long, and it cannot determine the inconsistency in the number of GAA repeat sequences on alleles, thus failing to accurately obtain allele results. While the more advanced second-generation sequencing technology has high accuracy, its high cost and complex data analysis process make it inefficient for testing small numbers of samples. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides a primer pair, kit, and method for detecting the FXN gene. These methods are convenient, time-efficient, and can accurately analyze the GAA repeat sequence number of the FXN gene.
[0006] The present invention adopts the following technical solution: When the FA-related FXN gene is pathogenic, more than 65 GAA repeat sequences will be amplified. Based on this, the present invention provides a primer pair, kit and method for detecting the number of GAA repeat sequences.
[0007] This invention provides a primer pair for detecting GAA repetitive sequences in the FXN gene, the sequences of which are as follows: FXN-F: 5'-CCACACGTGTTATTTGGCC-3' FXN-R: 5'-CATGGCCACACTTGCCT-3'.
[0008] Furthermore, the reverse primer FXN-R has a FAM fluorescent group at its 5' end.
[0009] The present invention also provides a kit for detecting GAA repetitive sequences in the FXN gene, comprising primer pairs FXN-F and FXN-R.
[0010] Furthermore, the PCR amplification system included in the kit comprises: 2ul of 10x PCR Buffer, 2ul of 2mM dNTPs, 4ul of 5x Q-solution, 0.1ul of 5 U / ul Taq DNA polymerase, 8.9ul of nuclease-free purified water, and 0.5ul of FXN-F and 0.5ul of the primer pair as described in claim 1 or 2.
[0011] This invention also provides a non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene, comprising: Genomic DNA is extracted from the sample to be tested and used as a DNA template; Configure a PCR amplification system containing the primer pair and the DNA template; DNA templates were amplified by PCR using primer pairs to obtain PCR products containing GAA trinucleotide repeat sequences; The PCR amplification products from step 3) were detected by capillary electrophoresis. The number of GAA trinucleotide repeats in the FXN gene of the sample to be tested was calculated based on the results of capillary electrophoresis.
[0012] Furthermore, the PCR amplification system includes: 2ul of 10x PCR Buffer, 2ul of 2mM dNTPs, 4ul of 5x Q-solution, 0.1ul of 5 U / ul Taq DNA polymerase, 8.9ul of nuclease-free purified water, and 0.5ul of primer pair FXN-F and 0.5ul of primer pair FXN-R.
[0013] Furthermore, the PCR amplification procedure includes: denaturing at 95°C for 15 minutes in a PCR instrument; then running the following cycle 40 times: denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extending at 72°C for 45 seconds; followed by extending at 72°C for 10 minutes and storing at 4°C.
[0014] Furthermore, the capillary electrophoresis in step 4 includes: diluting the PCR amplification product; mixing the calibration mixture containing a fluorescently labeled length standard with the PCR amplification product to be tested in a certain proportion to prepare a test mixture; and performing capillary electrophoresis detection on the test mixture.
[0015] The PCR amplification product was diluted 5 times with ddH2O; ABI GS600 LIZ internal standard and HIDI were mixed at a volume ratio of 1:50, and 9 μL of the mixture was mixed with 1 μL of the diluted PCR amplification product to prepare the instrumentation solution.
[0016] Furthermore, step 5) of calculating the GAA repeat status of the FXN gene includes: establishing a reference standard through sequencing verification, which defines the relationship between at least one known GAA repeat number and the corresponding DNA fragment length; using data analysis software to obtain the DNA fragment length of the target amplification product in capillary electrophoresis in the sample to be tested; and converting the obtained DNA fragment length into the corresponding GAA repeat sequence number based on the reference standard.
[0017] The present invention also provides the application of the primer pair for detecting GAA repetitive sequences of the FXN gene or the kit for detecting GAA repetitive sequences of the FXN gene in the detection of GAA repetitive sequences of the FXN gene.
[0018] The beneficial effects of this invention: 1. The primer pairs, reagent kits, and detection methods provided by this invention are easy to operate, technically mature, and can efficiently detect FXN pathogenic gene mutations with high accuracy and low cost. The capillary electrophoresis method of this invention is efficient and time-saving, making it very suitable for clinical testing. It solves the problems of long detection time and inability to determine allele results in first-generation sequencing, as well as the high cost of second-generation sequencing.
[0019] 2. The primers designed for the GAA repeat sequence of the FXN gene in this invention have high specificity. First-generation sequencing cannot determine the inconsistency in the number of GAA repeat sequences on alleles. The detection method of this invention can accurately and clearly determine this, and is less likely to cause non-specific amplification. In addition, since there is a continuous A base before the position of the GAA repeat sequence of the FXN gene in this invention, first-generation sequencing is very likely to produce double peaks, increasing the sequencing failure rate, which can easily lead to reduced personnel efficiency and increased costs.
[0020] 3. The GAA repetitive sequence of the FXN gene in FA patients can reach 65 times or even higher. First-generation sequencing is not suitable for detecting highly repetitive sequences, while capillary electrophoresis can adjust the detection range according to different liz internal standards.
[0021] 4. GeneMapper software was used for analysis. By setting up panels, markers, and bins using existing data, the capillary electrophoresis results were directly clear and easy to understand. Attached Figure Description
[0022] Figure 1 The capillary electrophoresis single-peak result of the reference standard sample indicates that the number of GAA repeat sequences on the alleles is the same.
[0023] Figure 2 The results of first-generation sequencing are used as a reference standard sample.
[0024] Figure 3 The capillary electrophoresis result of sample 3 indicates that the number of GAA repeats in the alleles is the same.
[0025] Figure 4 This is the first-generation sequencing result of sample 3 to be tested.
[0026] Figure 5 The capillary electrophoresis double peak result of sample 5 indicates that the number of GAA repeat sequences on the alleles is different.
[0027] Figure 6 This is the first-generation sequencing result of sample 5 to be tested.
[0028] Figure 7 This is the original sequence of the FXN gene in this invention, totaling 496 bp. The sequence contains 6 GAA repeat sequences, including primer pairs FXN-F and FXN-R, and the positions of the GAA repeat sequences. Detailed Implementation
[0029] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Example 1
[0030] Targeting the GAA repeat sequence of the FXN gene, such as Figure 7 As shown, a 496 bp fragment of the FXN gene was selected, containing 6 GAA repeat sequences. A specific primer pair was designed for this fragment. The reverse primer of the primer pair has a FAM fluorescent group at the 5' end. The primer pair sequence is as follows: FXN-F: 5'-CCACACGTGTTATTTGGCC-3' FXN-R: 5'-CATGGCCACACTTGCCT-3'. Example 2
[0031] Blood sample DNA extraction was performed using the Blood Genomic DNA Extraction Kit (DP348) from Tiangen Biotech (Beijing) Co., Ltd. 1. Add 200 μL of blood sample to a 1.5 ml centrifuge tube. 2. Add 20 μL of proteinase K solution and mix well.
[0032] 3. Add 200 μL of buffer GB, mix thoroughly by inverting, and incubate at 56 °C for 10 minutes, inverting and mixing several times during this period, until the solution becomes clear (if the solution is not completely clear, the lysis time needs to be extended until it becomes clear). Briefly centrifuge to remove water droplets from the inner wall of the tube cap.
[0033] 4. Add 200 μL of anhydrous ethanol and mix thoroughly by inverting. At this point, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the cap.
[0034] 5. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 back into the collection tube.
[0035] 6. Add 500 μL of buffer GB to the adsorption column CB3 (please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.
[0036] 7. Add 600 μL of PWB wash buffer to the adsorption column CB3 (please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.
[0037] 8. Repeat step 7 once.
[0038] 9. Centrifuge at 12,000 rpm (13,400 × g) for 2 minutes and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.
[0039] 10. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of elution buffer TB to the middle of the adsorption membrane, incubate at room temperature for 2-5 minutes, centrifuge at 12,000 rpm (13,400×g) for 2 minutes, and collect the solution into the centrifuge tube.
[0040] 11. Use Nano Drop 2000 to measure the concentration of the extracted DNA. If you are not going to continue to the next experiment, you need to store it at -20℃. Example 3
[0041] The blood sample DNA from Example 2 was amplified by PCR using the primer pair described in Example 1. The reverse primer FXN-R has a FAM fluorescent group at its 5' end. A commercially available PCR kit was used; in this example, the HotStarTaq DNA Polymerase (203203) kit from Qiagen, Germany was used. The procedure is as follows: 1. Reagent Preparation: Prepare the amplification reaction mixture according to the number of DNA samples to be tested, and dispense 20 μL per sample. The reagent quantities in the amplification reaction mixture are as follows:
[0042] Where N = number of samples to be tested + 1; Add 2 μL of DNA to 18 μL of amplification reaction mixture; Amplification: Perform PCR amplification in a PCR instrument. The amplification program is as follows: denature at 95℃ for 15 minutes in the PCR instrument; then run the following cycle 40 times: denature at 94℃ for 30 seconds, anneal at 58℃ for 30 seconds, extend at 72℃ for 45 seconds; then extend at 72℃ for 10 minutes, and store at 4℃. Example 4
[0043] The PCR products obtained in Example 3 were detected by capillary electrophoresis using a 3500xl sequencer, as follows: Sample dilution: Dilute the PCR product 5-fold by adding 10 μL of PCR amplification product to 40 μL ddH2O.
[0044] Prepare the PCR mixture: Mix ABI GS600-LIZ internal standard and HIDI at a volume ratio of 1:50, then mix 9 μL of the mixture with 1 μL of diluted PCR amplification product to prepare the PCR mixture.
[0045] 3. Capillary electrophoresis: The mixture was detected in an ABI 35000xl sequencer.
[0046] Example 5 After the 3500xl sequencer finished running the program, the capillary electrophoresis results were analyzed in GeneMapper software; and the accuracy of the CE results was verified and the reference standard for determining the number of GAA repeat sequences in the sample was determined using the first-generation sequencing results.
[0047] Example 6 Nine randomly selected test samples were analyzed according to the steps described in Examples 1 to 5 above. A reference standard sample (with nine repeats of GAA at 505 bp) was also added during the analysis. The analysis results of the same samples were compared using capillary electrophoresis and first-generation sequencing to evaluate the accuracy of this method.
[0048] The results of Example 6 are shown in Table 1. Verification showed that the number of GAA repeat sequences in the first-generation sequencing results of the 9 samples was the same as the number of GAA repeat sequences calculated from capillary electrophoresis for one allele, indicating that capillary electrophoresis has extremely high accuracy (error not exceeding one repeat). Conversely, CE results can also be used to verify the sequencing results.
[0049] Table 1 Comparison of first-generation sequencing results and capillary electrophoresis results
[0050] 1. Analysis of results based on the standard sample (Sample A): Figure 1 For example, the result of capillary electrophoresis is a single peak, and the fragment length is 504.60 bp. Figure 2 As shown, the corresponding sequencing result is a single peak with 9 GAA repeat sequences. After multiple CE single peaks and sequencing verification, the reference standard is that the number of GAA repeats at approximately 505bp is 9. Based on this, by setting panel-marker-bin in GeneMapper software, the number of GAA repeat sequences can be obtained intuitively.
[0051] The calculation method is as follows: taking the GAA repeat sequence at approximately 505 bp of the FXN gene as a reference standard of 9, the GAA repeat sequence of the FXN gene in the test sample is calculated. If a fluorescence signal peak appears at 505±3n bp of the FXN gene in the test sample, the number of GAA repeat sequences is 9±n.
[0052] 2. Alleles have the same GAA repeat sequence (CE results show only one peak). Taking the third experimental sample (hereinafter referred to as sample 3) as an example, if... Figure 3As shown, the CE result of sample 3 has only one peak at 505.80 bp, and the sequencing result of sample 3 also shows 9 GAA repeat sequences, as... Figure 4 As shown.
[0053] 3. Alleles have different GAA repeat sequences (CE results show two different peaks). Taking the position close to 505bp, which corresponds to GAA9, as a reference standard, the first peak corresponds to the GAA repeat number of the first allele. Fluorescence spurs appearing approximately every 3nbp before and after the first peak are selected, and the highest peak corresponds to the GAA repeat sequence number of the second allele.
[0054] Taking the fifth test sample (hereinafter referred to as sample 5) as an example, as follows: Figure 5 As shown, the CE results for sample 5 show the first allele at 501.68 bp and the second allele at 508.84 bp, indicating 8 and 10 GAA repeat sequences, respectively. However, the sequencing results for sample 5 only identified 10 GAA repeat sequences; the number of GAA repeats on the other allele could not be determined. Figure 6 As shown.
[0055] Therefore, compared to first-generation sequencing which cannot determine the inconsistency in the number of GAA repeat sequences on alleles, the detection method of the present invention can clearly determine the number of GAA repeat sequences in the FXN gene, with accurate results and convenient operation.
Claims
1. A primer pair for detecting GAA repetitive sequences in the FXN gene, characterized in that, The primer pair sequences are as follows: FXN-F: 5'-CCACACGTGTTATTTGGCC-3' FXN-R: 5'-CATGGCCACACTTGCCT-3'.
2. The primer pair for detecting GAA repetitive sequences in the FXN gene according to claim 1, characterized in that, The primer pair has a FAM fluorescent group at the 5' end of the reverse primer FXN-R.
3. A kit for detecting GAA repetitive sequences in the FXN gene, characterized in that, The kit contains the primer pair as described in claim 1 or 2.
4. The kit for detecting GAA repetitive sequences in the FXN gene according to claim 3, characterized in that, The PCR amplification system of the kit includes: 2ul of 10x PCR Buffer, 2ul of 2mM dNTPs, 4ul of 5x Q-solution, 0.1ul of 5 U / ul Taq DNA polymerase, 8.9ul of nuclease-free purified water, and 0.5ul of primer pair FXN-F and 0.5ul of primer pair FXN-R.
5. A non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene, characterized in that, include: Genomic DNA was extracted from the sample to be tested and used as a DNA template. Configure a PCR amplification system containing the primer pair as described in claim 1 or 2 and the DNA template of step 1); PCR amplification was performed on the DNA template from step 1) using primer pairs to obtain PCR amplification products containing GAA trinucleotide repeat sequences. The PCR amplification products from step 3) were detected by capillary electrophoresis. The GAA trinucleotide repeat sequence in the FXN gene of the sample to be tested was calculated based on the capillary electrophoresis results.
6. The non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene according to claim 5, characterized in that, The PCR amplification system includes: 2ul of 10x PCR Buffer, 2ul of 2mM dNTPs, 4ul of 5x Q-solution, 0.1ul of 5U / ul Taq DNA polymerase, 8.9ul of nuclease-free purified water, and 0.5ul of primer pair FXN-F and 0.5ul of primer pair FXN-R.
7. The non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene according to claim 5, characterized in that, The PCR amplification procedure includes: denaturing at 95°C for 15 minutes in a PCR instrument; then running the following cycle 40 times: denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extending at 72°C for 45 seconds; followed by extending at 72°C for 10 minutes and storing at 4°C.
8. The non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene according to claim 5, characterized in that, Step 4) of capillary electrophoresis includes: diluting the PCR amplification product; mixing the calibration mixture containing the fluorescently labeled length standard with the PCR amplification product to be tested in a certain proportion to prepare a test mixture; and performing capillary electrophoresis detection on the test mixture.
9. The non-diagnostic detection method for detecting GAA repetitive sequences in the FXN gene according to claim 5, characterized in that, Step 5) of calculating the GAA repeat sequence of the FXN gene includes: establishing a reference standard through sequencing verification, which defines the relationship between at least one known GAA repeat number and the corresponding DNA fragment length; using data analysis software to obtain the DNA fragment length of the target amplification product in the test sample in capillary electrophoresis; and converting the obtained DNA fragment length into the corresponding GAA repeat sequence number based on the reference standard.
10. The application of the primer pair for detecting GAA repetitive sequences of the FXN gene as described in claim 1 or the kit for detecting GAA repetitive sequences of the FXN gene as described in claim 3 in the detection of GAA repetitive sequences of the FXN gene.