CN105506107A - Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene - Google Patents
Primers and method for detecting polymorphic hotspot mutation condition of DOCK2 gene Download PDFInfo
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Abstract
The invention discloses primers and a method for detecting myelosis myelodysplastic syndrome and especially detecting DOCK2 gene mutation of a patient with myelosis myelodysplastic syndrome. The primers comprise (i) primers for amplifying 6th, 22nd, 33rd, 37th, 40th and 44th exon sequences of the DOCK2 gene. An Sanger sequencing technique and sequencing primers are adopted. The primers and method can be used for quickly detecting mutation of 6th, 22nd, 33rd, 37th, 40th and 44th exons of the DOCK2 gene in the body of a patient with myelosis myelodysplastic syndrome. The primers and method have the advantage of accurate detection result, can be used for assisted diagnosis of severe combined immunodeficiency disease, and have important reference meanings for early intervention and early therapy.
Description
Technical field
The invention belongs to life science and biological technical field, particularly detect primer and the method for DOCK2 gene polymorphic hot spot mutation situation.
Background technology
Combined immunodeficiency disease is a kind of disease of multifactorial inheritance, and it can be divided into: x joins heredity, autosomal recessive inheritance type and distributes type.Combined immunodeficiency disease will cause the congenital prosoplasia of lymphoid stem cells, T cell and B cell is lacked after baby due, humoral immunization and cellular immunization is made all defect to occur, the immunoglobulin (Ig) of oneself can not be synthesized, thus cause patient to irresistances such as bacterium, fungi, viruses, easily send out invasive infection.Infant is morbidity in 1-2 month after birth, as can not get effective treatment, more than dead in 1 years old.Abroad this disease is classified as " emergency case " at present, clearly carries out hematopoietic stem cell transplantation immediately once diagnose, can in time for infant wins new life.
DOCK2 gene is positioned on the mankind's No. 5 karyomit(e), overall length 446136bp, totally 52 exons.The protein of DOCK2 genes encoding belongs to CDM protein family.It is specific expressed in hematopoietic cell, mainly in peripheral blood leucocyte, and participates in the actin cytoskeleton reconstruct needed for lymphocytic migration by activation Rac.When DOCK2 gene is undergone mutation, the activation of Rac1 is impaired, and defect occurs for migration and being aggregated in T cell, B cell and NK cell of Actin muscle of chemokine induction.The mouse lacking this gene shows serious damage in lymphocytic migration with in guiding.People's reports such as recent Dobbs, it finds the allelic mutation of division of cytoplasm 2 gene (DOCK2) in the patient body of 5 combined immunodeficiency diseases, and wherein 3 patients are by Interferon, rabbit or DOCK2 expression treatment postnormalize.The catastrophe of prompting Dock2 is significant to combined immunodeficiency disease.To the abrupt climatic change of DOCK2 undoubtedly can to diagnosis morning of combined immunodeficiency disease and timely immunotherapy targeted autoantibody play an important role.
Summary of the invention
The object of this invention is to provide a kind of primer detecting DOCK2 gene polymorphic mutantional hotspot, adopt round pcr, can be used for the catastrophe in DOCK2 gene polymorphic site in rapid detection Patients With Myelodysplastic Syndrome body.The primer of described detection DOCK2 gene polymorphic hot spot mutation situation, comprising:
The primer of amplification DOCK2 gene, its base sequence is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG
Further, also comprise sequencing primer, its base sequence is:
The sequencing primer base sequence detecting DOCK2 gene is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG
Further, primer sequence DOCK2-1F and DOCK2-1R is the primer of amplification DOCK2 gene the 6th exon sequence, primer sequence DOCK2-2F and DOCK2-2R is the primer of amplification DOCK2 gene the 22nd exon sequence, primer sequence DOCK2-3F and DOCK2-3R is the primer of amplification DOCK2 gene the 33rd exon sequence, primer sequence DOCK2-4F and DOCK2-4R is the primer of amplification DOCK2 gene the 37th exon sequence, primer sequence DOCK2-5F and DOCK2-5R is the primer of amplification DOCK2 gene the 40th exon sequence, primer sequence DOCK2-6F and DOCK2-6R is the primer of amplification DOCK2 gene the 44th exon sequence.
Present invention also offers the method detecting DOCK2 gene the 6th, 22,33,37,40,44 exon catastrophe, comprise the following steps:
1. extracting blood/in DNA;
2. with the DNA extracted in pcr amplification step 1;
3. the amplified production in pair step 2 checks order;
4. pair sequencing result judges, determines whether DOCK2 gene undergos mutation;
Wherein pcr amplification primer is:
The primer of amplification DOCK2 gene, its base sequence is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG
Further, sequencing primer base sequence is:
The sequencing primer base sequence detecting DOCK2 gene is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG
Present invention also offers a kind of test kit detecting DOCK2 gene polymorphic mutational site, comprising:
(i) blood DNA extraction agent;
(ii) detection system pcr amplification reaction liquid;
(iii) check order system reagent;
Wherein pcr amplification reaction liquid primer is:
(I) increases the primer of DOCK2 gene, and its base sequence is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG
Further, sequencing primer base sequence is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG
Beneficial effect: the present invention devises the primer of amplification DOCK2 the 6th, 22,33,37,40,44 exon sequence.Adopt round pcr, construct stable amplification system.By reaction conditions such as adjustment primer concentration, annealing temperature etc., amplification efficiency can be made to reach best.The mutation type of DOCK2 gene is indefinite, therefore whole for described DOCK2 6,22,33,37,40,44 exon sequences can all increase out by primer of the present invention, also ensure to undergo mutation in the position, where of no matter these exons, all there will not be undetected situation.Fluorescence quantitative PCR method of comparing reduces cost and the difficulty of detection.Fluorescence quantitative PCR method will design multiple probe for different mutation types, cost is high, and detection difficulty is large.
Accompanying drawing explanation
Fig. 1 is DOCK2 gene location map on chromosome
Fig. 2 is the electrophorogram of DOCK2-1F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 2, primer DOCK2-1F/R increases effectively, and band is single.
Fig. 3 is the electrophorogram of DOCK2-2F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 3, primer DOCK2-2F/R increases effectively, and band is single.
Fig. 4 is the electrophorogram of DOCK2-3F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 4, primer DOCK2-3F/R increases effectively, and band is single.
Fig. 5 is the electrophorogram of DOCK2-4F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 5, primer DOCK2-4F/R increases effectively, and band is single.
Fig. 6 is the electrophorogram of DOCK2-5F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 6, primer DOCK2-5F/R increases effectively, and band is single.
Fig. 7 is the electrophorogram of DOCK2-6F/R, and No. 1-16, the blood sample of M to be MarkerDL2000,1-16 be censorship, as shown in Figure 7, primer DOCK2-6F/R increases effectively, and band is single.
Fig. 8,9,10,11,12,13 is respectively DOCK2 the 6th, 22,33,37,40, the 44 exon wild-type order-checking sectional drawing of sample 1,2,3,4,5,6.
Embodiment
Below in conjunction with specific embodiments and the drawings, set forth the present invention further.Should be noted that, unaccounted normal condition and method in embodiment, usually according to the conventional employing method of affiliated field experimenter: such as, " the fine works molecular biology experiment guide " the 4th edition of Ao Sibai and James Kingston chief editor, or the step of advising according to manufacturer and condition.
Embodiment 1
Detect the primer in DOCK2 gene polymorphic mutational site, the design of this primer is for the specificity amplification primer designed by DOCK2 mutantional hotspot, comprising:
Amplification DOCK2 gene comprises the primer of the 6th, 22,33,37,40,44 exon sequences, and its base sequence is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG
Detect the test kit in DOCK2 gene polymorphic mutational site, comprise
(i) blood DNA extraction agent;
(ii) detection system PCR reaction solution;
(iii) check order system reagent;
Wherein, tissue DNA extraction agent can purchased from commercial reagents such as sky root DNA extraction agent boxes.
Detection system pcr amplification reaction liquid comprises: 2 × PCRBuffer, 2mMdNTPs, KODFXDNAPolymerase (1U/ μ l), DOCK2 gene the 6th, 22, 33, 37, 40, 44 exon sequences upper, downstream primer DOCK2-1-F (10 μMs), DOCK2-1-R (10 μMs), DOCK2-2-F (10 μMs), DOCK2-2-R (10 μMs), DOCK2-3-F (10 μMs), DOCK2-3-R (10 μMs), DOCK2-4-F (10 μMs), DOCK2-4-R (10 μMs), DOCK2-5-F (10 μMs), DOCK2-5-R (10 μMs), DOCK2-6-F (10 μMs), DOCK2-6-R (10 μMs).
Order-checking system reagent comprises: order-checking refined solution (ExoI:0.6U, CIP:1.2U), EDTA (125mmol), dehydrated alcohol, 75% ethanol, HIDI (height deionized formamide), sequencing primer: the upstream and downstream primer detecting DOCK2 gene the 7th, 8,17 exon sequence is respectively M13-F (3.2 μm), M13-R (3.2 μm), and BigdyeTerminatorV3.1 (buying from AppliedBiosystems company of the U.S.).
Embodiment 2
The operating process of blood/cell/tissue genome DNA extraction test kit (sky root is biological):
(1) tissue DNA in extracting blood: 1) extract 300 μ l blood and add 900 μ l erythrocyte cracked liquids, put upside down mixing, room temperature places 5 minutes, and period puts upside down mixing several times again.The centrifugal 1min of 12,000rpm, sucks supernatant, leaves leukocyte cell pellet, adds 200 μ l damping fluid GA, and vibration is to thoroughly mixing.2) 20 μ l Proteinase K Solution are added, mixing.3) add 200 μ l damping fluid GB, fully put upside down mixing, place 10 minutes for 70 DEG C, solution strain is limpid, and brief centrifugation is to remove the globule of cap wall.4) add 200 μ l dehydrated alcohols, fully vibration mixing 15 seconds, now may occur flocks, brief centrifugation is to remove the globule of cap wall.5) all add in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, is put back in collection tube by adsorption column CB3.6) in adsorption column CB3, add 500 μ l damping fluid GD (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column CB3 is put into collection tube.7) in adsorption column CB3, add 700 μ l rinsing liquid PW (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column CB3 is put into collection tube.8) in adsorption column CB3, add 500 μ l rinsing liquid PW, centrifugal 30 seconds of 12,000rpm, outwells waste liquid.9) put back in collection tube by adsorption column CB3, centrifugal 2 minutes of 12,000rpm, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.10) proceeded to by adsorption column CB3 in a clean centrifuge tube, the unsettled dropping 100 in the middle part to adsorption film μ l elution buffer TE, room temperature places 2-5 minute, 12,000rpm centrifugal 2 minutes, by solution collection in centrifuge tube.
(2) reagent configuration: by detecting people's number configuration detection system PCR reaction solution each X μ l, every person-portion 19 μ l packing:
X=19 μ l reaction solution × (n part sample+1 part of blank)
N is for detecting number of samples.
(3) application of sample: add 1 μ lDNA in detection system PCR reaction solution; Blank adds 1 μ l physiological saline or does not add any material.
(4) increase: detect and carry out on Standard PCR instrument, available instrumentation comprises ABIveriti (AppliedBiosystems company of the U.S.) etc.Reaction conditions is as follows:
PCR amplification system preparation of reagents method is as follows:
Wherein, primer sequence is:
(5) electrophoresis: 1.5% agarose gel electrophoresis, 110V, 25min, gel imaging system is observed.
As shown in Fig. 2,3,4,5,6,7, be 16 routine blood samples with the electrophoretogram of products therefrom after DOCK2-1F/R, DOCK2-2F/R, DOCK2-3F/R, DOCK2-4F/R, DOCK2-5F/R, DOCK2-6F/R primer amplification.The fragment length of the present invention's amplification is respectively 441bp, 365bp, 437bp, 290bp, 571bp, 318bp, show DOCK2-1F/R, DOCK2-2F/R, DOCK2-3F/R, DOCK2-4F/R, DOCK2-5F/R, DOCK2-6F/R of the present invention amplification effectively by the analysis of electrophorogram, and band is single.
(6) Sanger order-checking:
Get 9 μ lPCR products and 2 μ l purification system.Purifying is carried out according to following program:
1 μ l purified product is mixed according to following system with upper and lower sequencing primer respectively:
Sequencing reaction program:
Precipitation link:
In the product completing sequencing reaction, add the EDTA of 2 μ l125mmol, leave standstill 5min; Add 15 μ l dehydrated alcohols, whirlpool mixes; The centrifugal 30min of 3700rpm; Be inverted centrifugal 15sec, add 50 μ l70% ethanol, whirlpool mixes; The centrifugal 15min of 3700rpm; Be inverted centrifugal 15sec, be placed on 95 DEG C of metal baths; Denatured test is carried out after adding 10 μ lHi-Di.Denaturation program:
After denaturation program terminates, upper sequenator (ABI3730) checks order.
(7) result judges: sequencing result and DOCK2 wild-type reference sequence (Genbankaccn:NC_000005.10) are compared respectively, report according to actual catastrophe to result.
Embodiment 3
The clinical sample (sample number is 1-16) getting 16 examples extracts genome, reagent preparation, amplification and order-checking by the reagent of embodiment 1 and 2 and method.Every increment originally adds 1 μ l in detection system PCR reaction solution.Electrophoresis result, as shown in Fig. 2,3,4,5,6,7, show that primer DOCK2-1F/R, DOCK2-2F/R, DOCK2-3F/R, DOCK2-4F/R, DOCK2-5F/R, DOCK2-6F/R of the present invention can effectively increase to blood sample, and band is single.
The detected result of sample 1,2,3,4,5,6 is as shown in Fig. 8,9,10,11,12,13:
Fig. 8 display is the DOCK26 exon wild-type order-checking sectional drawing of sample 1, illustrates that 6 exons of sample 1 are not undergone mutation.
Fig. 9 display is the DOCK222 exon wild-type order-checking sectional drawing of sample 2, illustrates that 22 exons of sample 2 are not undergone mutation.
Figure 10 display is the DOCK233 exon wild-type order-checking sectional drawing of sample 3, illustrates that 33 exons of sample 3 are not undergone mutation.
Figure 11 display is the DOCK237 exon wild-type order-checking sectional drawing of sample 4, illustrates that 37 exons of sample 4 are not undergone mutation.
Figure 12 display is the DOCK240 exon wild-type order-checking sectional drawing of sample 5, illustrates that 40 exons of sample 5 are not undergone mutation.
Figure 13 display is the DOCK244 exon wild-type order-checking sectional drawing of sample 6, illustrates that 44 exons of sample 6 are not undergone mutation.
As can be seen from detected result, primer of the present invention has been included exon sequence, can expand DOCK2 gene the 6th, 22,33,37,40,44 exons, and sequencing result entirely accurate.Primer of the present invention can expand accurately DOCK2 gene the 6th, 22,33,37,40,44 exons, no matter be wild-type or saltant type.The detection of positive sample is shown that primer of the present invention and method and test kit can detect DOCK2 transgenation.
Claims (6)
1. detect the primer of DOCK2 gene polymorphic hot spot mutation situation, it is characterized in that, comprising:
The primer of amplification DOCK2 gene, its base sequence is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG。
2. primer as claimed in claim 1, it is characterized in that, also comprise sequencing primer, its base sequence is:
The sequencing primer base sequence detecting DOCK2 gene is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG。
3. primer as claimed in claim 1, it is characterized in that, primer sequence DOCK2-1F and DOCK2-1R is the primer of amplification DOCK2 gene the 6th exon sequence, primer sequence DOCK2-2F and DOCK2-2R is the primer of amplification DOCK2 gene the 22nd exon sequence, primer sequence DOCK2-3F and DOCK2-3R is the primer of amplification DOCK2 gene the 33rd exon sequence, primer sequence DOCK2-4F and DOCK2-4R is the primer of amplification DOCK2 gene the 37th exon sequence, primer sequence DOCK2-5F and DOCK2-5R is the primer of amplification DOCK2 gene the 40th exon sequence, primer sequence DOCK2-6F and DOCK2-6R is the primer of amplification DOCK2 gene the 44th exon sequence.
4. primer as claimed in claim 2, is characterized in that, primer sequence M13-F and M13-R is the primer that order-checking DOCK2 gene amplification product comprises the 6th, 22,33,37,40,44 exon sequences.
5. detect the method for DOCK2 gene polymorphic hot spot mutation situation, comprise the following steps:
(1) tissue DNA in extracting blood;
(2) with the DNA extracted in pcr amplification step 1;
(3) amplified production in step 2 is checked order;
(4) sequencing result is judged, determine whether DOCK2 gene undergos mutation;
Wherein pcr amplification primer is:
DOCK2-1F：TGTAAAACGACGGCCAGTTTATGGGTTGTTTCTTGTCTCCT
DOCK2-1R：AACAGCTATGACCATGCCATCGGAAGTAGGTTAAATGAG
DOCK2-2F：TGTAAAACGACGGCCAGTCTTTAACCTTTGATTGAATGCCT
DOCK2-2R：AACAGCTATGACCATGTAGGTTGGATTCTTGAGCTGTTT
DOCK2-3F：TGTAAAACGACGGCCAGTCGTCGCCGAACTGTTTTAT
DOCK2-3R：AACAGCTATGACCATGGAGAAAGAGGCTAAGCAAATACA
DOCK2-4F：TGTAAAACGACGGCCAGTGTTATCCAGGGAGGAGGACTTT
DOCK2-4R：AACAGCTATGACCATGCTCTTCAACTTGCTGTGAGGC
DOCK2-5F：TGTAAAACGACGGCCAGTGCTTACTGGCTGCTTTACGAG
DOCK2-5R：AACAGCTATGACCATGTATGATGAGGGCTATTGACTGG
DOCK2-6F：TGTAAAACGACGGCCAGTTTGCAATATTTATTCTGCTTTCG
DOCK2-6R：AACAGCTATGACCATGGTCTCCTTTTTAAAGTCAAAGGG。
6. method as claimed in claim 5, it is characterized in that, sequencing primer base sequence is:
M13F：TGTAAAACGACGGCCAGT
M13R：AACAGCTATGACCATG。
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| CN106521012A (en) * | 2016-12-29 | 2017-03-22 | 天津协和华美医学诊断技术有限公司 | Detection reagent kit for detecting MDS (myelodysplastic syndrome)-related gene group |
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| CN109486938A (en) * | 2018-12-18 | 2019-03-19 | 武汉艾迪康医学检验所有限公司 | Detect method, primer and the application of SMN1 and SMN2 gene mutation |
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| CN109504755A (en) * | 2018-11-26 | 2019-03-22 | 武汉艾迪康医学检验所有限公司 | Detect primer, method and the kit of TSC2 gene point mutation |
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