CN113817816A - Primer, kit and method for detecting EPAS1 gene mutation - Google Patents
Primer, kit and method for detecting EPAS1 gene mutation Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a method for detecting EPAS1 gene variation, which comprise a primer for amplifying EPAS1 gene full exons; sanger sequencing technology was used. The invention can quickly detect the variation of the EPAS1 gene full exon. The detection result completed by the invention is accurate, and the invention can assist in diagnosing familial polycythemia (ECYT4), and has important reference significance for diagnosis and treatment of diseases.
Description
Technical Field
The invention belongs to the field of molecular detection, and particularly relates to a kit and a method for detecting EPAS1 gene mutation.
Background
Familial Erythrocytosis (Familial Erythrocystosis) is a rare autosomal dominant hereditary hematological disorder that may be congenital (e.g., hereditary) or acquired, and can be divided into primary and secondary. The abnormal structure of the erythrocyte membrane is caused by the defect of the erythrocyte membrane protein of the disease, so that the spherical erythrocytes in the blood are increased. The primary function of these cells is to transport oxygen from the lungs to tissues and organs throughout the body.
Symptoms and signs of familial polycythemia include headache, dizziness, nosebleed, and shortness of breath. Excessive red blood cells also increase the risk of abnormal blood clots that may block arterial and venous blood flow. If these clots restrict blood flow to vital organs and tissues (particularly the heart, lungs or brain), life-threatening complications such as heart attack or stroke can result. However, many familial polycythemia patients have only mild signs and symptoms, or never have any problems associated with their redundant red blood cells.
EPAS1 is called hypoxia inducible factor 2 alpha (HIF-2 alpha), it belongs to oxygen metabolism regulator, and participates in many vital activities of organism, plays an important role in many aspects such as in vivo energy metabolism, angiogenesis, inflammation, etc., and the research at present shows that EPAS1 gene mutation is related to familial type 4 of erythrocytosis, and belongs to secondary pathogenesis. The gene is located on chromosome 2, 16 exons. Hypoxia Inducible Factor (HIF) is a heterodimeric transcription factor composed of two protein subunits, namely alpha and beta, in 1997, endothelial PAS protein 1 (endoghial PASprotein-1, EPAS1) which is hypoxia inducible factor 2(HIF2 alpha) is cloned by Tian and the like, is a member of basic helix-loop-helix-PAS (bHLH-PAS) transcription factor superfamily, and comprises a bHLH domain, a PAS domain, a trans-activation domain and a nuclear entry signal. HIF2 α has many structural similarities to HIF1 α, with approximately 48% amino acid sequence homology. HIF2 α mRNA is highly expressed in vascular rich organs such as heart, lung, placenta, but is rarely expressed in leukocytes. HIF1 mRNA is detectable in all tissues of humans, but is not expressed at high levels in highly vascularized organs. HIF2 alpha regulates downstream target gene through Hypoxia Response Element (HREs), there is a DNA sequence < 100bp on the promoter or enhancer of its target gene, namely hypoxia response element, its core is 5 '-TACGTGCT-3' sequence. HIF2a binds to target genes through the recognition sequences of HREs, inducing transcription of the target genes, and thereby mediating cellular responses to hypoxia. The EPAS1 gene is expressed differently in different tissue cells of human or mouse. Under normal pressure, EPAS1 mRNA is present in high amounts in tissues such as lungs, heart, placenta, etc., which are rich in blood vessels, but is rarely expressed in leukocytes.
Studies of Van Xiaowei et al found that the sites rs75591953 and rs75984373 are related to reducing Han nationality plateau erythrocytosis (HAPC). In addition, the spleen is a main organ for destroying red blood cells, so that the method for clinically treating familial polycythemia by removing the spleen has a good curative effect, red blood cell membrane defects and spherical red blood cells still exist after the spleen is removed, the service life of the red blood cells is prolonged due to the removal of a main production place, and anemia is corrected, so that the early diagnosis of familial polycythemia is important, and the detection of a gene mutation site can predict diseases at the early stage of the onset.
The EPAS1 mutation analysis has important significance for familial polycythemia genetic consultation and prenatal diagnosis, and the genetic mode of ECYT4 is autosomal dominant inheritance. Therefore, if the disease-causing gene is clear, parents decide that the pregnancy should be subjected to relevant genetic counseling in time, prenatal molecular diagnosis is performed in the early stage of the pregnancy, and once an abnormal result occurs, whether treatment can be performed or not, how the treatment can be performed after the treatment and the like need to be clear, and feasible diagnosis and treatment measures are taken. Minimizing the birth of abnormal fetus and improving the population quality of newborn.
Disclosure of Invention
The invention aims to provide a kit for detecting EPAS1 gene mutation, which can be used for rapidly detecting the EPAS1 gene mutation in a patient by adopting a PCR (polymerase chain reaction) technology.
The primers for detecting EPAS1 gene mutation comprise primers for amplifying EPAS1 gene, and the base sequences of the primers are as follows:
further, the primers also comprise a sequencing primer for detecting the EPAS1 gene, and the base sequence of the sequencing primer is as follows:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG
further, the primers amplify corresponding exons as:
the invention also provides a method for detecting EPAS1 gene mutation, which comprises the following steps:
(1) extracting tissue DNA in blood;
(2) amplifying the DNA extracted in the step 1 by using PCR; wherein the PCR amplification primers are:
| EPAS1-1F | TGTAAAACGACGGCCAGTGCCCAGACGACCTCATAAAC |
| EPAS1-1R | AACAGCTATGACCATGTCTCAGCACTCTTCTCCCAA |
| EPAS1-2F | TGTAAAACGACGGCCAGTGGTCCCAGGCATTGGTAGT |
| EPAS1-2R | AACAGCTATGACCATGGCTGGTAAGGCTGGCATCT |
| EPAS1-3F | TGTAAAACGACGGCCAGTCATCCCTGGCAAATGCCTAT |
| EPAS1-3R | AACAGCTATGACCATGGCAGGGAGGAGACTGAATTG |
| EPAS1-4F | TGTAAAACGACGGCCAGTGAAGGTGGCTCAGCTTACTC |
| EPAS1-4R | AACAGCTATGACCATGCCTCTCAATGCCCCAATCTC |
| EPAS1-5-6F | TGTAAAACGACGGCCAGTCACAGGTGCTAAGAGAGCAG |
| EPAS1-5-6R | AACAGCTATGACCATGGGGCACTGATGCAATGAAAC |
| EPAS1-7F | TGTAAAACGACGGCCAGTTTGATTTGCCTTCTGGGGTT |
| EPAS1-7R | AACAGCTATGACCATGGGGCATAAGTCTTGTCTGCC |
| EPAS1-8F | TGTAAAACGACGGCCAGTCCTGGTCCTCACTGTCGT |
| EPAS1-8R | AACAGCTATGACCATGATCCCACTGTTCCAAGACATC |
| EPAS1-9F | TGTAAAACGACGGCCAGTGGTTTCCATGCATCTAGGGG |
| EPAS1-9R | AACAGCTATGACCATGGAGCGTGTGGTGTTCTTTTG |
| EPAS1-10F | TGTAAAACGACGGCCAGTAGGGAGTCTCTACGTTGACT |
| EPAS1-10R | AACAGCTATGACCATGGTTACTGCCCTGTATGGGGA |
| EPAS1-11F | TGTAAAACGACGGCCAGTGAACCTTTGGGTCCAGGAAG |
| EPAS1-11R | AACAGCTATGACCATGTACCCTGTACCCCTACCTCT |
| EPAS1-12F | TGTAAAACGACGGCCAGTAGGGGATAAACATGGGGGAA |
| EPAS1-12R | AACAGCTATGACCATGAGAGAAGGGCTAAATGGGGT |
| EPAS1-13-14F | TGTAAAACGACGGCCAGTGAGACTCTGCCTTTTGGGTCT |
| EPAS1-13-14R | AACAGCTATGACCATGGGTGAATGGAAGAACCAGGAT |
| EPAS1-15F | TGTAAAACGACGGCCAGTTCCCACACACCCAACTTTTC |
| EPAS1-15R | AACAGCTATGACCATGGAACTTGTGAGGAGGTTCCC |
| EPAS1-16F-1 | TGTAAAACGACGGCCAGTATTGGTATCCCCCAGTCACA |
| EPAS1-16R-1 | AACAGCTATGACCATGTTTAGGAAAAGCCACGCTGT |
| EPAS1-16F-2 | TGTAAAACGACGGCCAGTGTGAAGGGTCAACTCCAACG |
| EPAS1-16R-2 | AACAGCTATGACCATGGGACGGGGTCACTATACCAT |
| EPAS1-16F-3 | TGTAAAACGACGGCCAGTCCATGAGATGGTTTAGACGGG |
| EPAS1-16R-3 | AACAGCTATGACCATGACTTCACTTTACATTGGCATAGC |
(3) sequencing the amplification product in the step 2; the sequencing primer base sequence is:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG；
(4) and judging the sequencing result to determine whether the EPAS1 gene has mutation.
The invention finally provides a kit for detecting EPAS1 gene mutation sites, which comprises:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR amplification reaction solution; comprises a primer for amplifying an EPAS1 gene, and the base sequence of the primer is as follows:
| EPAS1-1F | TGTAAAACGACGGCCAGTGCCCAGACGACCTCATAAAC |
| EPAS1-1R | AACAGCTATGACCATGTCTCAGCACTCTTCTCCCAA |
| EPAS1-2F | TGTAAAACGACGGCCAGTGGTCCCAGGCATTGGTAGT |
| EPAS1-2R | AACAGCTATGACCATGGCTGGTAAGGCTGGCATCT |
| EPAS1-3F | TGTAAAACGACGGCCAGTCATCCCTGGCAAATGCCTAT |
| EPAS1-3R | AACAGCTATGACCATGGCAGGGAGGAGACTGAATTG |
| EPAS1-4F | TGTAAAACGACGGCCAGTGAAGGTGGCTCAGCTTACTC |
| EPAS1-4R | AACAGCTATGACCATGCCTCTCAATGCCCCAATCTC |
| EPAS1-5-6F | TGTAAAACGACGGCCAGTCACAGGTGCTAAGAGAGCAG |
| EPAS1-5-6R | AACAGCTATGACCATGGGGCACTGATGCAATGAAAC |
| EPAS1-7F | TGTAAAACGACGGCCAGTTTGATTTGCCTTCTGGGGTT |
| EPAS1-7R | AACAGCTATGACCATGGGGCATAAGTCTTGTCTGCC |
| EPAS1-8F | TGTAAAACGACGGCCAGTCCTGGTCCTCACTGTCGT |
| EPAS1-8R | AACAGCTATGACCATGATCCCACTGTTCCAAGACATC |
| EPAS1-9F | TGTAAAACGACGGCCAGTGGTTTCCATGCATCTAGGGG |
| EPAS1-9R | AACAGCTATGACCATGGAGCGTGTGGTGTTCTTTTG |
| EPAS1-10F | TGTAAAACGACGGCCAGTAGGGAGTCTCTACGTTGACT |
| EPAS1-10R | AACAGCTATGACCATGGTTACTGCCCTGTATGGGGA |
| EPAS1-11F | TGTAAAACGACGGCCAGTGAACCTTTGGGTCCAGGAAG |
| EPAS1-11R | AACAGCTATGACCATGTACCCTGTACCCCTACCTCT |
| EPAS1-12F | TGTAAAACGACGGCCAGTAGGGGATAAACATGGGGGAA |
| EPAS1-12R | AACAGCTATGACCATGAGAGAAGGGCTAAATGGGGT |
| EPAS1-13-14F | TGTAAAACGACGGCCAGTGAGACTCTGCCTTTTGGGTCT |
| EPAS1-13-14R | AACAGCTATGACCATGGGTGAATGGAAGAACCAGGAT |
| EPAS1-15F | TGTAAAACGACGGCCAGTTCCCACACACCCAACTTTTC |
| EPAS1-15R | AACAGCTATGACCATGGAACTTGTGAGGAGGTTCCC |
| EPAS1-16F-1 | TGTAAAACGACGGCCAGTATTGGTATCCCCCAGTCACA |
| EPAS1-16R-1 | AACAGCTATGACCATGTTTAGGAAAAGCCACGCTGT |
| EPAS1-16F-2 | TGTAAAACGACGGCCAGTGTGAAGGGTCAACTCCAACG |
| EPAS1-16R-2 | AACAGCTATGACCATGGGACGGGGTCACTATACCAT |
| EPAS1-16F-3 | TGTAAAACGACGGCCAGTCCATGAGATGGTTTAGACGGG |
| EPAS1-16R-3 | AACAGCTATGACCATGACTTCACTTTACATTGGCATAGC |
(iii) sequencing system reagents; comprises a sequencing primer for detecting EPAS1 gene, and the base sequence of the sequencing primer is as follows:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
has the advantages that: the invention designs a primer for amplifying EPAS1 exon sequences 1-16. The PCR products of each of the 16 pairs of primers can be sequenced with one sequencing primer by adding a linker. A stable amplification system is constructed by adopting a PCR technology. By adjusting the reaction conditions such as primer concentration and annealing temperature, the amplification efficiency can be optimized. The EPAS1 gene has various mutation types and is distributed throughout the whole gene, so the primer can amplify the EPAS1 exon sequences and ensure that the condition of omission does not occur no matter where the exons are mutated. Compared with a fluorescent quantitative PCR method, the method reduces the cost and difficulty of detection. The fluorescent quantitative PCR method needs to design a plurality of probes aiming at different mutation types, and has high cost and great detection difficulty.
Drawings
FIG. 1 is a map of the location of the EPAS1 gene on a chromosome.
FIG. 2 shows the electrophoresis of exon 1-16 primers, M is Marker DL 2000, and as shown, the primers are all amplified efficiently with bright bands.
Figure 3 is a representation of the sequencing of exon 2 of EPAS 1.
Figure 4 is a representation of the sequencing of exon 6 of EPAS 1.
Figure 5 is a representation of the sequencing of exon 16 of EPAS 1.
Detailed Description
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.
Example 1
A primer for detecting EPAS1 gene mutation sites, which is designed to be an amplification primer designed aiming at EPAS1 exon and comprises the following components:
the primer for amplifying the full exon sequence of the EPAS1 gene has the base sequence as follows:
a kit for detecting mutations in EPAS1 gene, comprising:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR reaction solution;
(iii) sequencing system reagents;
the tissue DNA extraction reagent can be purchased from commercial reagents such as Tiangen DNA extraction kit and the like.
The PCR amplification reaction solution of the detection system comprises: 2 times PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U/. mu.l); the primer concentrations of the upstream primer and the downstream primer of the exon sequence of the EPAS1 gene are both 10. mu.M.
The sequencing system reagent comprises: sequencing purification solution (ExoI:0.6U, CIP:1.2U), EDTA (125mmol), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: the upstream and downstream primers for detecting the exon sequences of EPAS1 gene were M13-F (3.2 μ M), M13-R (3.2 μ M), and Bigdye Terminator V3.1 (purchased from Applied Biosystems, USA), respectively.
Example 2
The operation flow of the blood/cell/tissue genome DNA extraction kit (Tiangen organism):
(1) extracting tissue DNA from blood: 1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuge at 12,000 rpm for 1min, aspirate the supernatant, leave the leukocyte pellet, add 200. mu.l of buffer GA, and shake until thoroughly mixed. 2) Add 20. mu.l proteinase K solution and mix well. 3) Add 200. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap. 4) Add 200. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover. 5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging at 12,000 rpm for 30 s, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe. 6) Add 500. mu.l buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, dump the waste and place adsorption column CB3 in the collection tube. 7) To the adsorption column CB3, 700. mu.l of a rinsing solution PW (previously used, whether or not absolute ethyl alcohol has been added) was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. 8) To the adsorption column CB3, 500. mu.l of a rinsing solution PW was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. 9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. 10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000 rpm, and collecting the solution into the centrifuge tube.
(2) Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the parts of detected people, and subpackaging 19 mul of PCR reaction liquid of each part of detected people:
19. mu.l reaction solution X (n parts specimen +1 part blank control)
And n is the number of detected samples.
(3) Sample adding: adding 1 mul DNA into the PCR reaction solution of the detection system; blank control was supplemented with 1. mu.l of physiological saline or nothing.
(4) Amplification: the detection is carried out on a conventional PCR instrument, and available instruments include ABI veriti (Applied Biosystems, USA) and the like. The reaction conditions were as follows:
the preparation method of the PCR amplification system reagent comprises the following steps:
wherein, the primer sequence is as follows:
(5) electrophoresis: electrophoresis on 1.5% agarose gel at 110V for 25min, and observation on a gel imaging system.
As shown in FIG. 2, the electrophoresis pattern of the product obtained after the blood sample is amplified by using EPAS1-1F \ R, EPAS1-2F \ R, EPAS1-3F \ R, EPAS1-5-6F \ R, EPAS1-7F \ R, EPAS1-8F \ R, EPAS1-9F \ R, EPAS1-10F \ R, EPAS1-11F \ R, EPAS1-12F \ R, EPAS1-13-14F \ R, EPAS1-15F \ R, EPAS1-16-1F \ R, EPAS1-16-2F \ R, EPAS1-16-3F \ R as the primer. The length of the amplified fragment is 915, 548, 570, 334, 744, 338, 473, 472, 645, 368, 843, 714, 530, 885, 691 and 893bp, and analysis of an electrophoretogram shows that the primers are amplified effectively and have single bands.
(6) Sanger sequencing:
take 9. mu.l of PCR product and 2. mu.l of purification system. Purification was performed according to the following procedure:
mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:
reaction procedure:
and (3) a precipitation link:
adding 2 mu l of 125mmol EDTA into the product after the sequencing reaction, and standing for 5 min; adding 15 mul of absolute ethyl alcohol, and mixing evenly by vortex; centrifuging at 3700rpm for 30 min; inverting, centrifuging for 15sec, adding 50 μ l 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; denaturation test was performed after adding 10. mu.l Hi-Di.
After the denaturation procedure was completed, sequencing was performed using a sequencer (ABI 3730).
(7) And (5) judging a result: the sequencing results were compared with the EPAS1 wild-type reference sequence, respectively
(Genbank: NG-016000.1) and the results are reported in terms of actual mutations.
Example 3
1 clinical sample was taken and genome extraction, reagent formulation, amplification and sequencing were performed according to the reagents and methods of examples 1 and 2. Mu.l of sample was added to each PCR reaction solution. The electrophoresis result is shown in FIG. 2, the band is single and bright, which indicates that the primers EPAS1-1F \ R, EPAS1-2F \ R, EPAS1-3F \ R, EPAS1-5-6F \ R, EPAS1-7F \ R, EPAS1-8F \ R, EPAS1-9F \ R, EPAS1-10F \ R, EPAS1-11F \ R, EPAS1-12F \ R, EPAS1-13-14F \ R, EPAS1-15F \ R, EPAS1-16-1F \ R, EPAS1-16-2F R, EPAS1-16-3F \ R are effective for blood sample amplification.
As can be seen from the analysis of the sequencing results (see FIGS. 3 and 4), the primers of the present invention have included all exons, can amplify the whole exons of the EPAS1 gene, and have accurate sequencing results.
Sequence listing
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<400> 22
tgtaaaacga cggccagtag gggataaaca tgggggaa 38
<210> 23
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aacagctatg accatgagag aagggctaaa tggggt 36
<210> 24
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tgtaaaacga cggccagtga gactctgcct tttgggtct 39
<210> 25
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
aacagctatg accatgggtg aatggaagaa ccaggat 37
<210> 26
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tgtaaaacga cggccagttc ccacacaccc aacttttc 38
<210> 27
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
aacagctatg accatggaac ttgtgaggag gttccc 36
<210> 28
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tgtaaaacga cggccagtat tggtatcccc cagtcaca 38
<210> 29
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aacagctatg accatgttta ggaaaagcca cgctgt 36
<210> 30
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgtaaaacga cggccagtgt gaagggtcaa ctccaacg 38
<210> 31
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
aacagctatg accatgggac ggggtcacta taccat 36
<210> 32
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tgtaaaacga cggccagtcc atgagatggt ttagacggg 39
<210> 33
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
aacagctatg accatgactt cactttacat tggcatagc 39
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tgtaaaacga cggccagt 18
<210> 34
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
aacagctatg accatg 16
Claims (4)
1. The primer for detecting EPAS1 gene variation is characterized by comprising a primer for amplifying the whole exon of EPAS1 gene, and the base sequence of the primer is as follows:
EPAS1-1F： TGTAAAACGACGGCCAGTGCCCAGACGACCTCATAAAC
EPAS1-1R： AACAGCTATGACCATGTCTCAGCACTCTTCTCCCAA
EPAS1-2F： TGTAAAACGACGGCCAGTGGTCCCAGGCATTGGTAGT
EPAS1-2R： AACAGCTATGACCATGGCTGGTAAGGCTGGCATCT
EPAS1-3F： TGTAAAACGACGGCCAGTCATCCCTGGCAAATGCCTAT
EPAS1-3R： AACAGCTATGACCATGGCAGGGAGGAGACTGAATTG
EPAS1-4F： TGTAAAACGACGGCCAGTGAAGGTGGCTCAGCTTACTC
EPAS1-4R： AACAGCTATGACCATGCCTCTCAATGCCCCAATCTC
EPAS1-5-6F： TGTAAAACGACGGCCAGTCACAGGTGCTAAGAGAGCAG
EPAS1-5-6R： AACAGCTATGACCATGGGGCACTGATGCAATGAAAC
EPAS1-7F： TGTAAAACGACGGCCAGTTTGATTTGCCTTCTGGGGTT
EPAS1-7R： AACAGCTATGACCATGGGGCATAAGTCTTGTCTGCC
EPAS1-8F： TGTAAAACGACGGCCAGTCCTGGTCCTCACTGTCGT
EPAS1-8R： AACAGCTATGACCATGATCCCACTGTTCCAAGACATC
EPAS1-9F： TGTAAAACGACGGCCAGTGGTTTCCATGCATCTAGGGG
EPAS1-9R： AACAGCTATGACCATGGAGCGTGTGGTGTTCTTTTG
EPAS1-10F： TGTAAAACGACGGCCAGTAGGGAGTCTCTACGTTGACT
EPAS1-10R： AACAGCTATGACCATGGTTACTGCCCTGTATGGGGA
EPAS1-11F： TGTAAAACGACGGCCAGTGAACCTTTGGGTCCAGGAAG
EPAS1-11R： AACAGCTATGACCATGTACCCTGTACCCCTACCTCT
EPAS1-12F： TGTAAAACGACGGCCAGTAGGGGATAAACATGGGGGAA
EPAS1-12R： AACAGCTATGACCATGAGAGAAGGGCTAAATGGGGT
EPAS1-13-14F： TGTAAAACGACGGCCAGTGAGACTCTGCCTTTTGGGTCT
EPAS1-13-14R： AACAGCTATGACCATGGGTGAATGGAAGAACCAGGAT
EPAS1-15F： TGTAAAACGACGGCCAGTTCCCACACACCCAACTTTTC
EPAS1-15R： AACAGCTATGACCATGGAACTTGTGAGGAGGTTCCC
EPAS1-16F-1： TGTAAAACGACGGCCAGTATTGGTATCCCCCAGTCACA
EPAS1-16R-1： AACAGCTATGACCATGTTTAGGAAAAGCCACGCTGT
EPAS1-16F-2： TGTAAAACGACGGCCAGTGTGAAGGGTCAACTCCAACG
EPAS1-16R-2： AACAGCTATGACCATGGGACGGGGTCACTATACCAT
EPAS1-16F-3： TGTAAAACGACGGCCAGTCCATGAGATGGTTTAGACGGG
EPAS1-16R-3： AACAGCTATGACCATGACTTCACTTTACATTGGCATAGC。
2. the primers as claimed in claim 1, further comprising a sequencing primer for detecting the EPAS1 gene, which has the base sequence:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
3. a method for detecting EPAS1 gene mutation, comprising the following steps:
(1) extracting genome DNA in blood;
(2) amplifying the DNA extracted in the step 1 by using PCR; wherein the PCR amplification primers are:
EPAS1-1F： TGTAAAACGACGGCCAGTGCCCAGACGACCTCATAAAC
EPAS1-1R： AACAGCTATGACCATGTCTCAGCACTCTTCTCCCAA
EPAS1-2F： TGTAAAACGACGGCCAGTGGTCCCAGGCATTGGTAGT
EPAS1-2R： AACAGCTATGACCATGGCTGGTAAGGCTGGCATCT
EPAS1-3F： TGTAAAACGACGGCCAGTCATCCCTGGCAAATGCCTAT
EPAS1-3R： AACAGCTATGACCATGGCAGGGAGGAGACTGAATTG
EPAS1-4F： TGTAAAACGACGGCCAGTGAAGGTGGCTCAGCTTACTC
EPAS1-4R： AACAGCTATGACCATGCCTCTCAATGCCCCAATCTC
EPAS1-5-6F： TGTAAAACGACGGCCAGTCACAGGTGCTAAGAGAGCAG
EPAS1-5-6R： AACAGCTATGACCATGGGGCACTGATGCAATGAAAC
EPAS1-7F： TGTAAAACGACGGCCAGTTTGATTTGCCTTCTGGGGTT
EPAS1-7R： AACAGCTATGACCATGGGGCATAAGTCTTGTCTGCC
EPAS1-8F： TGTAAAACGACGGCCAGTCCTGGTCCTCACTGTCGT
EPAS1-8R： AACAGCTATGACCATGATCCCACTGTTCCAAGACATC
EPAS1-9F： TGTAAAACGACGGCCAGTGGTTTCCATGCATCTAGGGG
EPAS1-9R： AACAGCTATGACCATGGAGCGTGTGGTGTTCTTTTG
EPAS1-10F： TGTAAAACGACGGCCAGTAGGGAGTCTCTACGTTGACT
EPAS1-10R： AACAGCTATGACCATGGTTACTGCCCTGTATGGGGA
EPAS1-11F： TGTAAAACGACGGCCAGTGAACCTTTGGGTCCAGGAAG
EPAS1-11R： AACAGCTATGACCATGTACCCTGTACCCCTACCTCT
EPAS1-12F： TGTAAAACGACGGCCAGTAGGGGATAAACATGGGGGAA
EPAS1-12R： AACAGCTATGACCATGAGAGAAGGGCTAAATGGGGT
EPAS1-13-14F： TGTAAAACGACGGCCAGTGAGACTCTGCCTTTTGGGTCT
EPAS1-13-14R： AACAGCTATGACCATGGGTGAATGGAAGAACCAGGAT
EPAS1-15F： TGTAAAACGACGGCCAGTTCCCACACACCCAACTTTTC
EPAS1-15R： AACAGCTATGACCATGGAACTTGTGAGGAGGTTCCC
EPAS1-16F-1： TGTAAAACGACGGCCAGTATTGGTATCCCCCAGTCACA
EPAS1-16R-1： AACAGCTATGACCATGTTTAGGAAAAGCCACGCTGT
EPAS1-16F-2： TGTAAAACGACGGCCAGTGTGAAGGGTCAACTCCAACG
EPAS1-16R-2： AACAGCTATGACCATGGGACGGGGTCACTATACCAT
EPAS1-16F-3： TGTAAAACGACGGCCAGTCCATGAGATGGTTTAGACGGG
EPAS 1-16R-3: AACAGCTATGACCATGACTTCACTTTACATTGGCATAGC (3) sequencing the amplification product of step 2; the sequencing primer base sequence is:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG；
(4) and comparing the sequencing result with the normal sequence to determine whether the EPAS1 gene has variation.
4. Kit for detecting EPAS1 gene mutation sites, which is characterized in that the kit comprises:
(i) blood DNA extraction reagent;
(ii) detecting a system PCR amplification reaction solution; comprises a primer for amplifying an EPAS1 gene, and the base sequence of the primer is as follows:
EPAS1-1F： TGTAAAACGACGGCCAGTGCCCAGACGACCTCATAAAC
EPAS1-1R： AACAGCTATGACCATGTCTCAGCACTCTTCTCCCAA
EPAS1-2F： TGTAAAACGACGGCCAGTGGTCCCAGGCATTGGTAGT
EPAS1-2R： AACAGCTATGACCATGGCTGGTAAGGCTGGCATCT
EPAS1-3F： TGTAAAACGACGGCCAGTCATCCCTGGCAAATGCCTAT
EPAS1-3R： AACAGCTATGACCATGGCAGGGAGGAGACTGAATTG
EPAS1-4F： TGTAAAACGACGGCCAGTGAAGGTGGCTCAGCTTACTC
EPAS1-4R： AACAGCTATGACCATGCCTCTCAATGCCCCAATCTC
EPAS1-5-6F： TGTAAAACGACGGCCAGTCACAGGTGCTAAGAGAGCAG
EPAS1-5-6R： AACAGCTATGACCATGGGGCACTGATGCAATGAAAC
EPAS1-7F： TGTAAAACGACGGCCAGTTTGATTTGCCTTCTGGGGTT
EPAS1-7R： AACAGCTATGACCATGGGGCATAAGTCTTGTCTGCC
EPAS1-8F： TGTAAAACGACGGCCAGTCCTGGTCCTCACTGTCGT
EPAS1-8R： AACAGCTATGACCATGATCCCACTGTTCCAAGACATC
EPAS1-9F： TGTAAAACGACGGCCAGTGGTTTCCATGCATCTAGGGG
EPAS1-9R： AACAGCTATGACCATGGAGCGTGTGGTGTTCTTTTG
EPAS1-10F： TGTAAAACGACGGCCAGTAGGGAGTCTCTACGTTGACT
EPAS1-10R： AACAGCTATGACCATGGTTACTGCCCTGTATGGGGA
EPAS1-11F： TGTAAAACGACGGCCAGTGAACCTTTGGGTCCAGGAAG
EPAS1-11R： AACAGCTATGACCATGTACCCTGTACCCCTACCTCT
EPAS1-12F： TGTAAAACGACGGCCAGTAGGGGATAAACATGGGGGAA
EPAS1-12R： AACAGCTATGACCATGAGAGAAGGGCTAAATGGGGT
EPAS1-13-14F： TGTAAAACGACGGCCAGTGAGACTCTGCCTTTTGGGTCT
EPAS1-13-14R： AACAGCTATGACCATGGGTGAATGGAAGAACCAGGAT
EPAS1-15F： TGTAAAACGACGGCCAGTTCCCACACACCCAACTTTTC
EPAS1-15R： AACAGCTATGACCATGGAACTTGTGAGGAGGTTCCC
EPAS1-16F-1： TGTAAAACGACGGCCAGTATTGGTATCCCCCAGTCACA
EPAS1-16R-1： AACAGCTATGACCATGTTTAGGAAAAGCCACGCTGT
EPAS1-16F-2： TGTAAAACGACGGCCAGTGTGAAGGGTCAACTCCAACG
EPAS1-16R-2： AACAGCTATGACCATGGGACGGGGTCACTATACCAT
EPAS1-16F-3： TGTAAAACGACGGCCAGTCCATGAGATGGTTTAGACGGG
EPAS1-16R-3： AACAGCTATGACCATGACTTCACTTTACATTGGCATAGC
(iii) sequencing system reagents; comprises a sequencing primer for detecting EPAS1 gene, and the base sequence of the sequencing primer is as follows:
M13 F：TGTAAAACGACGGCCAGT
M13 R：AACAGCTATGACCATG。
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| CN103911380A (en) * | 2013-02-06 | 2014-07-09 | 深圳华大基因科技有限公司 | EPAS1 gene mutant and application thereof |
| WO2015126557A1 (en) * | 2014-02-24 | 2015-08-27 | Vanderbilt University | Identification of cattle at risk of high altitude pulmonary hypertension |
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| US5695963A (en) * | 1997-01-17 | 1997-12-09 | Board Of Regents, The University Of Texas System | Endothelial PAS domain protein |
| US20110171633A1 (en) * | 2010-01-11 | 2011-07-14 | Wayne Cowens | Method to use gene expression to determine likelihood of clinical outcome of renal cancer |
| CN103911380A (en) * | 2013-02-06 | 2014-07-09 | 深圳华大基因科技有限公司 | EPAS1 gene mutant and application thereof |
| WO2015126557A1 (en) * | 2014-02-24 | 2015-08-27 | Vanderbilt University | Identification of cattle at risk of high altitude pulmonary hypertension |
| CN109593841A (en) * | 2018-12-08 | 2019-04-09 | 南京艾迪康医学检验所有限公司 | Detect method, kit, oligonucleotides and its application of F13A1 gene mutation |
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