CN109593841A - Detect method, kit, oligonucleotides and its application of F13A1 gene mutation - Google Patents
Detect method, kit, oligonucleotides and its application of F13A1 gene mutation Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses the primers and method of a kind of F13A1 gene mutation for detecting coagulation factor deficiency patient comprising the primer of the amplification full exon sequence of F13A1 gene；Using Sanger sequencing technologies and sequencing primer.The present invention can rapidly come out the abrupt climatic change of the full exon of coagulation factor deficiency patient's body F13A1 gene.The testing result completed using the present invention is accurate, can be with auxiliary diagnosis coagulation factor deficiency, and the specific aim of risk profile and therapeutic scheme to disease is custom made with important reference significance.
Description
Technical field
The invention belongs to bioscience and field of biotechnology, in particular to a kind of method of F13A1 gene mutation, reagent
Box, oligonucleotides and its application.
Background technique
Coagulation factor is a kind of protein component for participating in Blood Coagulation Process.Their physiological action is, in angiorrbagia
When be activated, together with platelet adhesion, to block the leak on blood vessel.According to it is found that coagulation factor it is successive suitable
Sequence, coagulation factor are named as coagulation factor 1~13, and wherein coagulation factor 13 is the last one factor for participating in coagulation process,
Activity with transglutaminase plays the role of stable fibers fibrin clot.The shortage of coagulation factor 13 will lead to blood coagulation
Block is unstable.
Coagulation factor 13 is not covalently linked by two pairs of different peptide chains, these peptide chains are respectively by F13A1 and F13B base
Because of coding.F13A1 and F13B gene is located on different chromosome, and wherein F13A1 is located at 6p24-25, overall length 160kb,
Containing 15 exons and 14 intrones, 731 amino acid are encoded, the mutation occurred on F13A1 gene is genetic solidifying
The principal pathogenetic reason of 13 deficiency disease of blood factor.
13 deficiency disease of hereditary coagulation factors is a kind of rare autosomal recessive hereditary diseases, disease incidence only about 1/
2000000, male to female ratio is about 1:1.The main clinical manifestation of this disease is lifelong hemorrhagic tendency, poor wound healing and habit
Inertia miscarriage etc..Since nineteen sixty Duckert etc. reports the first patient, more than 300 examples of report are amounted to, the country only finds numerical example.
The presently found gene mutation about more than 70 for leading to 13 deficiency disease of coagulation factor, wherein most mutation occur in F13A1 base
Because upper.Compared with mutation occurs in 13 deficiency disease of coagulation factor on F13B gene, the blood coagulation on F13A1 gene occurs for mutation
Clinical symptoms caused by 13 deficiency disease of the factor are more serious.Since 13 deficiency disease patient's bleeding of hereditary coagulation factors shows individual
It differs greatly, serious visceral hemorrhage concurrent infection often occurs after microtrauma for some individuals, while coagulation factor 13 lacks
Disease routine blood coagulation inspection and platelet count and function are normal, therefore are easy wrong diagnosis and escape, so carrying out to suspected patient
The full exon sequencing of F13A1 helps to make a definite diagnosis early, this is to the prevention severe haemorrhage of later period immunotherapy targeted autoantibody without being suspected to have important meaning
Justice.
Summary of the invention
The purpose of the present invention is to provide the oligonucleotides of detection F13A1 gene mutation, using round pcr, can be used for fast
The F13A1 gene extron catastrophe of speed detection 13 deficiency disease patient's body of coagulation factor.
The present invention provides the oligonucleotides of detection F13A1 gene mutation, comprising: at least outside pair for amplification F13A1 gene
The primer of son is shown, at least pair for amplification primer is selected from F13A1-1F/F13A1-1R, F13A1-2F/F13A1-2R, F13A1-
3F/F13A1-3R、F13A1-4F/F13A1-4R、F13A1-5F/F13A1-5R、F13A1-6F/F13A1-6R、F13A1-7F/
F13A1-7R、F13A1-8F/F13A1-8R、F13A1-9F/F13A1-9R、F13A1-10F/F13A1-10R、F13A1-11F/
F13A1-11R、F13A1-12F/F13A1-12R、F13A1-13F/F13A1-13R、F13A1-14F/F13A1-14R、F13A1-
15-1F/F13A1-15-1R or F13A1-15-2F/F13A1-15-2R, base sequence are as follows:
F13A1-1F:TGTAAAACGACGGCCAGTCCAGCAACTGGTTGCGGT；
F13A1-1R:AACAGCTATGACCATGCCCCATTTGGTGGCATTCTA
F13A1-2F:TGTAAAACGACGGCCAGTTGCTGTAGGATTATGGAGGGTG；
F13A1-2R:AACAGCTATGACCATGGGCAGAACTGGGACAAGGCT；
F13A1-3F:TGTAAAACGACGGCCAGTAGTGCCTTGTGGTCTGAAACG；
F13A1-3R:AACAGCTATGACCATGACTGTGCCTGTACCCACCTCT；
F13A1-4F:TGTAAAACGACGGCCAGTTGCAGACTTGCCTGATTTGTAT；
F13A1-4R:AACAGCTATGACCATGACCTCCAACTCCCGAACTCA；
F13A1-5F:TGTAAAACGACGGCCAGTAAAGAGCCAGAGTTCGTCAGC；
F13A1-5R:AACAGCTATGACCATGCGCAGTTGTCTTTATGAGTCCC；
F13A1-6F:TGTAAAACGACGGCCAGTGGCCTGACTTTGGTAAATGATT；
F13A1-6R:AACAGCTATGACCATGACTGGCATATATACTGAGGCAAA；
F13A1-7F:TGTAAAACGACGGCCAGTTGGTAAACCGAACAGAAGTGG；
F13A1-7R:AACAGCTATGACCATGAATTCTATAGTGTCAACAGGGGC；
F13A1-8F:TGTAAAACGACGGCCAGTAACCCTGTTGGGATTCTACTCTA；
F13A1-8R:AACAGCTATGACCATGGTTTTCTGCCTGTGCTGTTGA；
F13A1-9F:TGTAAAACGACGGCCAGTGCCTGGCCGTCACTTCTT；
F13A1-9R:AACAGCTATGACCATGCTCCTGTCTTCAAAACTGCTTAG；
F13A1-10F:TGTAAAACGACGGCCAGTTCTTCAGCCTTGGAAAGCATC；
F13A1-10R:AACAGCTATGACCATGTGTGTTAAGAGGTTGGGGAGAA；
F13A1-11F:TGTAAAACGACGGCCAGTCAAGGACAGAGGAGGGAGAAA；
F13A1-11R:AACAGCTATGACCATGAAATGCTGCAAATGCCAGTG；
F13A1-12F:TGTAAAACGACGGCCAGTGTGCAGTACACGGTGCATCC；
F13A1-12R:AACAGCTATGACCATGAATGAGTGGGTTCTCCCTGTG；
F13A1-13F:TGTAAAACGACGGCCAGTCTTTACCTAAGAAGCAGGACAGC；
F13A1-13R:AACAGCTATGACCATGTAAGTCTGCGTGCCTTTGTCT；
F13A1-14F:TGTAAAACGACGGCCAGTAGACAACAAGAGAGCAGAACGAG；
F13A1-14R:AACAGCTATGACCATGAATGGTCGGCAAGGAGGAG；
F13A1-15-1F:TGTAAAACGACGGCCAGTTCTTCAGTGTCCTAAGCACCAAC；
F13A1-15-1R:AACAGCTATGACCATGGGCCACCTCTTCTAACAAGGTA；
F13A1-15-2F:TGTAAAACGACGGCCAGTTTTTCCCAGGACTAGAGCACAA；
F13A1-15-2R:AACAGCTATGACCATGTACTGCGGAGGAAGGATGGT。
Further, the oligonucleotides further includes a pair of of sequencing primer, base sequence are as follows:
M13F:TGTAAAACGACGGCCAGT；
M13R:AACAGCTATGACCATG.
Further, F13A1-1F:F13A1-1R, F13A1-2F:F13A1-2R, F13A1-3F:F13A1-3R, F13A1-
4F:F13A1-4R, F13A1-5F:F13A1-5R, F13A1-6F:F13A1-6R, F13A1-7F:F13A1-7R, F13A1-8F:
F13A1-8R, F13A1-9F:F13A1-9R, F13A1-10F:F13A1-10R, F13A1-11F:F13A1-11R, F13A1-12F:
F13A1-12R, F13A1-13F:F13A1-13R, F13A1-14F:F13A1-14R, F13A1-15-1F:F13A1-15-1R and
The ratio of F13A1-15-2F:F13A1-15-2R is 1.
Further, the ratio of M13F:M13R is 1.
Application of the oligonucleotides provided by the invention in preparation detection coagulation factor deficiency kit.
The present invention also provides a kind of methods for detecting F13A1 gene mutation, comprising the following steps:
(1) genomic DNA in sample is extracted；
(2) it is expanded using the DNA at least pair for amplification primer pair (1), obtains amplified production；
(3) amplified production in (2) is sequenced using a pair of sequencing primer M13F and M13R, obtains the amplification and produces
The base sequence of object；
(4) base sequence in (3) is compared with F13A1 gene wild-type reference sequence, determines that mutational site is
No presence；Wherein at least pair for amplification primer is selected from F13A1-1F/F13A1-1R, F13A1-2F/F13A1-2R, F13A1-
3F/F13A1-3R、F13A1-4F/F13A1-4R、F13A1-5F/F13A1-5R、F13A1-6F/F13A1-6R、F13A1-7F/
F13A1-7R、F13A1-8F/F13A1-8R、F13A1-9F/F13A1-9R、F13A1-10F/F13A1-10R、F13A1-11F/
F13A1-11R、F13A1-12F/F13A1-12R、F13A1-13F/F13A1-13R、F13A1-14F/F13A1-14R、F13A1-
15-1F/F13A1-15-1R or F13A1-15-2F/F13A1-15-2R.
The present invention also provides a kind of kits for detecting F13A1 gene mutation, and the kit includes detection architecture PCR
Amplification reaction solution, sequencing system reaction solution, the detection architecture pcr amplification reaction liquid includes described in claim 1 at least one
To amplimer, the sequencing system reaction solution includes a pair of of sequencing primer as claimed in claim 2.
Further, the detection architecture pcr amplification reaction liquid further includes 2 × PCR Buffer, dNTPs and KOD FX
DNA Polymerase。
Further, the sequencing system reaction solution further includes EDTA, dehydrated alcohol, 75% ethyl alcohol, HIDI and Bigdye
Terminator V3.1。
Further, the sequencing system reaction solution further includes sequencing refined solution, and the sequencing refined solution includes outside nucleic acid
Enzyme cutting I and calf intestinal alkaline phosphatase.
The utility model has the advantages that the amplimer that (1) present invention designs all is added to M13 connector, make the PCR of all 16 pairs of primers
Product can be used a pair of of sequencing primer and is sequenced, and enormously simplify sequencing steps, reduce the fussy degree of detection；
(2) 16 pairs of amplimers provided by the invention cover all 15 exons of F13A1 gene, can guarantee no matter these are outer aobvious
The case where where position of son mutates, and is all not in missing inspection, especially to the 15th exon, it has longer sequence
It arranges, a part of sequence therein is usually only related in existing detection, and the present invention designs two pairs of primers, expands outside the 15th respectively
Two different pieces for showing son, to cover entire 15th exon；(3) primer that the present invention designs is dense by adjusting primer
The methods of degree, reaches amplification efficiency most preferably, and consistent annealing temperature can be used in all primers, has reduced detection
Difficulty；(4) it needs compared to common fluorescence quantitative PCR method for known possibility in F13A1 gene all 15 exons
Multiple probes are designed (assuming that each exon will then design 45 there are 3 mutational sites in the numerous mutational sites to mutate
Probe, testing cost dramatically increase), and unknown mutation can not be detected, present invention only requires (the 16 pairs of amplifications of 17 pairs of primers
Primer and 1 pair of sequencing primer) it can be detected, not only testing cost is significantly reduced, but also can detecte out unknown mutation.
Detailed description of the invention
Fig. 1 is the positioning figure of F13A1 gene on chromosome.
Fig. 2 is the amplified production electrophoretogram of 16 pairs of primers.Marker is DL2000.
Fig. 3,4,5,6,7,8,9,10,11,12,13,14,15 and 16 be respectively sample F13A1 gene the 1st, 2,3,4,5,
6, screenshot is sequenced in 7,8,9,10,11,12,13,14 exons.
Figure 17 and 18 is respectively that the first part of the 15th exon of sample F13A1 gene and the sequencing of second part are cut
Figure.
Specific embodiment
Combined with specific embodiments below and attached drawing, the present invention is further explained.It should be noted that not specified in embodiment
Normal condition and method, usually routinely use method according to fields experimenter: for example, Ao Sibai and James Kingston chief editor
" fine works molecular biology experiment guide " fourth edition, or according to step proposed by manufacturer and condition.
Embodiment 1
A kind of oligonucleotides detecting F13A1 gene polymorphic mutational site, base sequence are as follows:
F13A1-1F:TGTAAAACGACGGCCAGTCCAGCAACTGGTTGCGGT；
F13A1-1R:AACAGCTATGACCATGCCCCATTTGGTGGCATTCTA
F13A1-2F:TGTAAAACGACGGCCAGTTGCTGTAGGATTATGGAGGGTG；
F13A1-2R:AACAGCTATGACCATGGGCAGAACTGGGACAAGGCT；
F13A1-3F:TGTAAAACGACGGCCAGTAGTGCCTTGTGGTCTGAAACG；
F13A1-3R:AACAGCTATGACCATGACTGTGCCTGTACCCACCTCT；
F13A1-4F:TGTAAAACGACGGCCAGTTGCAGACTTGCCTGATTTGTAT；
F13A1-4R:AACAGCTATGACCATGACCTCCAACTCCCGAACTCA；
F13A1-5F:TGTAAAACGACGGCCAGTAAAGAGCCAGAGTTCGTCAGC；
F13A1-5R:AACAGCTATGACCATGCGCAGTTGTCTTTATGAGTCCC；
F13A1-6F:TGTAAAACGACGGCCAGTGGCCTGACTTTGGTAAATGATT；
F13A1-6R:AACAGCTATGACCATGACTGGCATATATACTGAGGCAAA；
F13A1-7F:TGTAAAACGACGGCCAGTTGGTAAACCGAACAGAAGTGG；
F13A1-7R:AACAGCTATGACCATGAATTCTATAGTGTCAACAGGGGC；
F13A1-8F:TGTAAAACGACGGCCAGTAACCCTGTTGGGATTCTACTCTA；
F13A1-8R:AACAGCTATGACCATGGTTTTCTGCCTGTGCTGTTGA；
F13A1-9F:TGTAAAACGACGGCCAGTGCCTGGCCGTCACTTCTT；
F13A1-9R:AACAGCTATGACCATGCTCCTGTCTTCAAAACTGCTTAG；
F13A1-10F:TGTAAAACGACGGCCAGTTCTTCAGCCTTGGAAAGCATC；
F13A1-10R:AACAGCTATGACCATGTGTGTTAAGAGGTTGGGGAGAA；
F13A1-11F:TGTAAAACGACGGCCAGTCAAGGACAGAGGAGGGAGAAA；
F13A1-11R:AACAGCTATGACCATGAAATGCTGCAAATGCCAGTG；
F13A1-12F:TGTAAAACGACGGCCAGTGTGCAGTACACGGTGCATCC；
F13A1-12R:AACAGCTATGACCATGAATGAGTGGGTTCTCCCTGTG；
F13A1-13F:TGTAAAACGACGGCCAGTCTTTACCTAAGAAGCAGGACAGC；
F13A1-13R:AACAGCTATGACCATGTAAGTCTGCGTGCCTTTGTCT；
F13A1-14F:TGTAAAACGACGGCCAGTAGACAACAAGAGAGCAGAACGAG；
F13A1-14R:AACAGCTATGACCATGAATGGTCGGCAAGGAGGAG；
F13A1-15-1F:TGTAAAACGACGGCCAGTTCTTCAGTGTCCTAAGCACCAAC；
F13A1-15-1R:AACAGCTATGACCATGGGCCACCTCTTCTAACAAGGTA；
F13A1-15-2F:TGTAAAACGACGGCCAGTTTTTCCCAGGACTAGAGCACAA；
F13A1-15-2R:AACAGCTATGACCATGTACTGCGGAGGAAGGATGGT；
The oligonucleotides further includes a pair of of sequencing primer M13F and M13R, base sequence are as follows:
M13F:TGTAAAACGACGGCCAGT；
M13R:AACAGCTATGACCATG.
A kind of kit detecting F13A1 gene mutation, including detection architecture pcr amplification reaction liquid and sequencing system reaction
Liquid, wherein
Detection architecture pcr amplification reaction liquid includes: 2 × PCR Buffer (10.0 μ L)；dNTPs(2mM)；KOD FX DNA
Polymerase(1U/μl)；The upstream and downstream primers F 13A1-1F (10 μM), F13A1-1R (10 μ of 15 exons of F13A1 gene
M)、F13A1-2F(10μM)、F13A1-2R(10μM)、F13A1-3F(10μM)、F13A1-3R(10μM)、F13A1-4F(10μ
M)、F13A1-4R(10μM)、F13A1-5F(10μM)、F13A1-5R(10μM)、F13A1-6F(10μM)、F13A1-6R(10μ
M)、F13A1-7F(10μM)、F13A1-7R(10μM)、F13A1-8F(10μM)、F13A1-8R(10μM)、F13A1-9F(10μ
M)、F13A1-9R(10μM)、F13A1-10F(10μM)、F13A1-10R(10μM)、F13A1-11F(10μM)、F13A1-11R
(10μM)、F13A1-12F(10μM)、F13A1-12R(10μM)、F13A1-13F(10μM)、F13A1-13R(10μM)、F13A1-
14F(10μM)、F13A1-14R(10μM)、F13A1-15-1F(10μM)、F13A1-15-1R(10μM)、F13A1-15-2F(10μ
M)、F13A1-15-2R(10μM)。
Sequencing system reaction solution include: sequencing refined solution (exonuclease I:0.6U, calf intestinal alkaline phosphatase:
1.2U)；EDTA(125mM)；Dehydrated alcohol；75% ethyl alcohol；HIDI (height deionized formamide)；A pair of of sequencing primer M13F
(3.2μM),M13R(3.2μM)；Bigdye Terminator V3.1 (is bought from Applied Biosystems company, the U.S.).
The kit may also include blood DNA extraction agent.
The kit may also include positive reference substance, negative controls and blank control product.Positive reference substance be containing
The solution of people's F13A1 gene extron DNA, forbids multigelation.Negative controls are ddH2O.Blank control product are 2 μ l physiology
Any substance is not added in salt water.
2 blood sample DNA of embodiment extracting
Blood sample DNA extracts (according to Tiangeng biological blood/cell/tissue gene DNA extracts kit specification): taking out
Human blood sample DNA is mentioned, specific method for extracting is as follows:
(1) 300 μ l blood are extracted and 900 μ l erythrocyte cracked liquids is added, be mixed by inversion, be placed at room temperature for 5 minutes, during which run again
It mixes several times.12000rpm is centrifuged 1min, sucks supernatant, leaves leukocyte cell pellet, adds 200 μ l buffer GA, vibrates to thorough
Bottom mixes；
(2) 20 μ l Proteinase K Solutions are added, mix；
(3) be added 200 μ l buffer GB, be sufficiently mixed by inversion, 70 DEG C place 10 minutes, solution strain it is limpid, briefly from
The heart is to remove the droplet of cap wall；
(4) 200 μ l dehydrated alcohols are added, sufficiently oscillation mixes 15 seconds, at this time it is possible that flocculent deposit, brief centrifugation
To remove the droplet of cap wall；
(5) previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12000rpm is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put back in collecting pipe；
(6) 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12000rpm is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put into collecting pipe；
(7) 700 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12000rpm is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put into collecting pipe；
(8) 500 μ l rinsing liquid PW, 12000rpm are added into adsorption column CB3 centrifugation 30 seconds, outwell waste liquid；
(9) adsorption column CB3 is put back in collecting pipe, 12000rpm is centrifuged 2 minutes, outwells waste liquid, adsorption column CB3 is placed in
It is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
(10) adsorption column CB3 is transferred in a clean centrifuge tube, 100 μ l is vacantly added dropwise to the intermediate position of adsorbed film
Elution buffer TE is placed at room temperature for 2~5 minutes, and 12000rpm is centrifuged 2 minutes, and solution is collected into centrifuge tube.
The amplification of 3 sample DNA of embodiment
According to the blood sample DNA that embodiment 2 extracts, people F13A 1 then is expanded using the amplimer in embodiment 1
Gene extron obtains amplified production.It is carried out on Standard PCR instrument when amplification, can include (the U.S. ABI veriti with instrument
Applied Biosystems company) etc..It notes at foot:
(i) sample number n (sample number=1+positive control of number of awaiting test sample+negative control, 1+blank control 1) is pressed
Survey system pcr amplification reaction liquid is taken, every 19 μ l of pipe is sub-packed in reaction tube；
(ii) 1 μ l is respectively taken to be separately added into reaction the above-mentioned sample to be tested handled well and negative controls, positive reference substance
Guan Zhong is mixed, the low-speed centrifugal several seconds, carries out PCR amplification, obtains amplified production.Test system pcr amplification reaction liquid making method
As shown in table 1.PCR amplification amplification reaction condition is as shown in table 2.The details of each pair of amplimer are as shown in table 3.
1. test system pcr amplification reaction liquid of table is prepared
Note: PrimerF and PrimerR in table be selected from F13A1-1F/F13A1-1R, F13A1-2F/F13A1-2R,
F13A1-3F/F13A1-3R、F13A1-4F/F13A1-4R、F13A1-5F/F13A1-5R、F13A1-6F/F13A1-6R、
F13A1-7F/F13A1-7R、F13A1-8F/F13A1-8R、F13A1-9F/F13A1-9R、F13A1-10F/F13A1-10R、
F13A1-11F/F13A1-11R、F13A1-12F/F13A1-12R、F13A1-13F/F13A1-13R、F13A1-14F/F13A1-
14R、F13A1-15-1F/F13A1-15-1R、F13A1-15-2F/F13A1-15-2R。
Table 2.PCR expands amplification reaction condition
Each pair of amplimer information of table 3.
4 Sanger of embodiment sequencing
Take the sequencing refined solution in the amplified production and 2 μ l embodiments 1 in 9 μ l embodiments 3 according to program described in table 4
It is purified, to obtain purified product.
4 purifying procedure of table
By 1 μ l purified product obtained respectively with sequencing primer M13F (3.2 μM), the M13R (3.2 μM) in embodiment 1
It is mixed according to the system in table 5, is then sequenced according to the sequencing reaction program in table 6.
Table 5
6. sequencing reaction program of table
Precipitate link:
The EDTA of 2 μ l 125mM is added into the product for completing sequencing reaction, stands 5min；15 μ l dehydrated alcohols are added,
Whirlpool mixes；3700rpm is centrifuged 30min；It is inverted centrifugation 15sec, 50 μ l, 70% ethyl alcohol is added, whirlpool mixes；3700rpm from
Heart 15min；It is inverted centrifugation 15sec, is placed on 95 DEG C of metal baths；Denatured test is carried out after 10 μ l HIDI are added.Denatured test step
Suddenly are as follows: 95 DEG C, 5min；Then it in -30 DEG C of 2min, is then saved at 4 DEG C.
After denaturation program, upper sequenator (ABI3730) sequencing.
As a result judge:
Sequencing result and F13A1 wild-type reference sequence (Genbank accn:NC_000006.12) are compared respectively
It is right, result is reported according to practical catastrophe.
The detection of 5 clinical sample of embodiment
Take clinical blood sample successively according to embodiment 1, embodiment 2, embodiment 3 and embodiment 4, reagent preparation, extracting
Sample DNA, amplification (obtaining amplified production) and sequencing.1 μ l of sample is added in detection architecture PCR reaction solution.
Using amplified production obtained, carry out DNA electrophoresis, deposition condition is 1.5% agarose gel electrophoresis, 110V,
25min, gel imaging system observation.Electrophoresis result as shown in Fig. 2, wherein 1-16 respectively correspond F13A1-1F/F13A1-1R,
F13A1-2F/F13A1-2R、F13A1-3F/F13A1-3R、F13A1-4F/F13A1-4R、F13A1-5F/F13A1-5R、
F13A1-6F/F13A1-6R、F13A1-7F/F13A1-7R、F13A1-8F/F13A1-8R、F13A1-9F/F13A1-9R、
F13A1-10F/F13A1-10R、F13A1-11F/F13A1-11R、F13A1-12F/F13A1-12R、F13A1-13F/F13A1-
The amplification of 13R, F13A1-14F/F13A1-14R, F13A1-15-1F/F13A1-15-1R, F13A1-15-2F/F13A1-15-2R
The electrophorogram of product, M is Marker DL 2000, by electrophoretogram analysis shows this 16 pairs of primer amplifications are effective, and item
With single.
The sequencing result of sample is as shown in Fig. 3~18.
Fig. 3 show be the 1st exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 1 extra of sample is aobvious
Son does not mutate.
Fig. 4 show be the 2nd exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 2 extras of sample are aobvious
Son does not mutate.
Fig. 5 show be the 3rd exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 3 extras of sample are aobvious
Son does not mutate.
Fig. 6 show be the 4th exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 4 extras of sample are aobvious
Son does not mutate.
Fig. 7 show be the 5th exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 5 extras of sample are aobvious
Son does not mutate.
Fig. 8 show be the 6th exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 6 extras of sample are aobvious
Son does not mutate.
Fig. 9 show be the 7th exon of F13A1 gene of sample wild type sequencing screenshot, illustrate that 7 extras of sample are aobvious
Son does not mutate.
Figure 10 show be sample the 8th exon of F13A1 gene wild type sequencing screenshot, illustrate 8 extras of sample
Aobvious son does not mutate.
Figure 11 show be sample the 9th exon of F13A1 gene wild type sequencing screenshot, illustrate 9 extras of sample
Aobvious son does not mutate.
Figure 12 show be sample the 10th exon of F13A1 gene wild type sequencing screenshot, illustrate No. 10 of sample
Exon does not mutate.
Figure 13 show be sample F13A1 gene o.11 exon wild type sequencing screenshot, illustrate No. 11 of sample
Exon does not mutate.
Figure 14 show be sample the 12nd exon of F13A1 gene wild type sequencing screenshot, illustrate No. 12 of sample
Exon does not mutate.
Figure 15 show be sample the 13rd exon of F13A1 gene wild type sequencing screenshot, illustrate No. 13 of sample
Exon does not mutate.
Figure 16 show be sample the 14th exon of F13A1 gene wild type sequencing screenshot, illustrate No. 14 of sample
Exon does not mutate.
Figure 17 show be sample the 15th exon first part of F13A1 gene wild type sequencing screenshot, illustrate sample
This 15 exon first parts do not mutate.
Figure 18 show be sample the 15th exon second part of F13A1 gene wild type sequencing screenshot, illustrate sample
This 15 exon second parts do not mutate.
From testing result as can be seen that primer of the present invention is F13A1 gene, totally 15 exon sequences include
Inside, F13A1 gene all 15 exons, and sequencing result entirely accurate can be expanded.By testing result it is found that
15 exons in sample are wild type.In addition, also indicating that this to the detection of F13A gene extron mutation positive control
Oligonucleotides, method and the kit of invention are capable of detecting when the catastrophe of F13A1 gene extron mutation.
Sequence table
<110>Co., Ltd, Nanjing Ai Dikang medical test institute
<120>method, kit, oligonucleotides and its application of F13A1 gene mutation are detected
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgtaaaacga cggccagtcc agcaactggt tgcggt 36
<210> 2
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aacagctatg accatgcccc atttggtggc attcta 36
<210> 3
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgtaaaacga cggccagttg ctgtaggatt atggagggtg 40
<210> 4
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aacagctatg accatgggca gaactgggac aaggct 36
<210> 5
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgtaaaacga cggccagtag tgccttgtgg tctgaaacg 39
<210> 6
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aacagctatg accatgactg tgcctgtacc cacctct 37
<210> 7
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tgtaaaacga cggccagttg cagacttgcc tgatttgtat 40
<210> 8
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aacagctatg accatgacct ccaactcccg aactca 36
<210> 9
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tgtaaaacga cggccagtaa agagccagag ttcgtcagc 39
<210> 10
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aacagctatg accatgcgca gttgtcttta tgagtccc 38
<210> 11
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tgtaaaacga cggccagtgg cctgactttg gtaaatgatt 40
<210> 12
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aacagctatg accatgactg gcatatatac tgaggcaaa 39
<210> 13
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tgtaaaacga cggccagttg gtaaaccgaa cagaagtgg 39
<210> 14
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aacagctatg accatgaatt ctatagtgtc aacaggggc 39
<210> 15
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tgtaaaacga cggccagtaa ccctgttggg attctactct a 41
<210> 16
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aacagctatg accatggttt tctgcctgtg ctgttga 37
<210> 17
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
tgtaaaacga cggccagtgc ctggccgtca cttctt 36
<210> 18
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
aacagctatg accatgctcc tgtcttcaaa actgcttag 39
<210> 19
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tgtaaaacga cggccagttc ttcagccttg gaaagcatc 39
<210> 20
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aacagctatg accatgtgtg ttaagaggtt ggggagaa 38
<210> 21
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
tgtaaaacga cggccagtca aggacagagg agggagaaa 39
<210> 22
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
aacagctatg accatgaaat gctgcaaatg ccagtg 36
<210> 23
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tgtaaaacga cggccagtgt gcagtacacg gtgcatcc 38
<210> 24
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
aacagctatg accatgaatg agtgggttct ccctgtg 37
<210> 25
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
tgtaaaacga cggccagtct ttacctaaga agcaggacag c 41
<210> 26
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
aacagctatg accatgtaag tctgcgtgcc tttgtct 37
<210> 27
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
tgtaaaacga cggccagtag acaacaagag agcagaacga g 41
<210> 28
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
aacagctatg accatgaatg gtcggcaagg aggag 35
<210> 29
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
tgtaaaacga cggccagttc ttcagtgtcc taagcaccaa c 41
<210> 30
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
aacagctatg accatgggcc acctcttcta acaaggta 38
<210> 31
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
tgtaaaacga cggccagttt ttcccaggac tagagcacaa 40
<210> 32
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
aacagctatg accatgtact gcggaggaag gatggt 36
<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 (10)
1. detecting the oligonucleotides of F13A1 gene mutation characterized by comprising at least pair for amplification F13A1 gene extron
Primer, at least pair for amplification primer be selected from F13A1-1F/F13A1-1R,
F13A1-2F/F13A1-2R、F13A1-3F/F13A1-3R、F13A1-4F/F13A1-4R、
F13A1-5F/F13A1-5R、F13A1-6F/F13A1-6R、F13A1-7F/F13A1-7R、
F13A1-8F/F13A1-8R、F13A1-9F/F13A1-9R、F13A1-10F/F13A1-10R、
F13A1-11F/F13A1-11R、F13A1-12F/F13A1-12R、F13A1-13F/F13A1-13R、
F13A1-14F/F13A1-14R, F13A1-15-1F/F13A1-15-1R, or
F13A1-15-2F/F13A1-15-2R, base sequence are as follows:
F13A1-1F:TGTAAAACGACGGCCAGTCCAGCAACTGGTTGCGGT；
F13A1-1R:AACAGCTATGACCATGCCCCATTTGGTGGCATTCTA
F13A1-2F:TGTAAAACGACGGCCAGTTGCTGTAGGATTATGGAGGGTG；
F13A1-2R:AACAGCTATGACCATGGGCAGAACTGGGACAAGGCT；
F13A1-3F:TGTAAAACGACGGCCAGTAGTGCCTTGTGGTCTGAAACG；
F13A1-3R:AACAGCTATGACCATGACTGTGCCTGTACCCACCTCT；
F13A1-4F:TGTAAAACGACGGCCAGTTGCAGACTTGCCTGATTTGTAT；
F13A1-4R:AACAGCTATGACCATGACCTCCAACTCCCGAACTCA；
F13A1-5F:TGTAAAACGACGGCCAGTAAAGAGCCAGAGTTCGTCAGC；
F13A1-5R:AACAGCTATGACCATGCGCAGTTGTCTTTATGAGTCCC；
F13A1-6F:TGTAAAACGACGGCCAGTGGCCTGACTTTGGTAAATGATT；
F13A1-6R:AACAGCTATGACCATGACTGGCATATATACTGAGGCAAA；
F13A1-7F:TGTAAAACGACGGCCAGTTGGTAAACCGAACAGAAGTGG；
F13A1-7R:AACAGCTATGACCATGAATTCTATAGTGTCAACAGGGGC；
F13A1-8F:TGTAAAACGACGGCCAGTAACCCTGTTGGGATTCTACTCTA；
F13A1-8R:AACAGCTATGACCATGGTTTTCTGCCTGTGCTGTTGA；
F13A1-9F:TGTAAAACGACGGCCAGTGCCTGGCCGTCACTTCTT；
F13A1-9R:AACAGCTATGACCATGCTCCTGTCTTCAAAACTGCTTAG；
F13A1-10F:TGTAAAACGACGGCCAGTTCTTCAGCCTTGGAAAGCATC；
F13A1-10R:AACAGCTATGACCATGTGTGTTAAGAGGTTGGGGAGAA；
F13A1-11F:TGTAAAACGACGGCCAGTCAAGGACAGAGGAGGGAGAAA；
F13A1-11R:AACAGCTATGACCATGAAATGCTGCAAATGCCAGTG；
F13A1-12F:TGTAAAACGACGGCCAGTGTGCAGTACACGGTGCATCC；
F13A1-12R:AACAGCTATGACCATGAATGAGTGGGTTCTCCCTGTG；
F13A1-13F:TGTAAAACGACGGCCAGTCTTTACCTAAGAAGCAGGACAGC；
F13A1-13R:AACAGCTATGACCATGTAAGTCTGCGTGCCTTTGTCT；
F13A1-14F:TGTAAAACGACGGCCAGTAGACAACAAGAGAGCAGAACGAG；
F13A1-14R:AACAGCTATGACCATGAATGGTCGGCAAGGAGGAG；
F13A1-15-1F:TGTAAAACGACGGCCAGTTCTTCAGTGTCCTAAGCACCAAC；
F13A1-15-1R:AACAGCTATGACCATGGGCCACCTCTTCTAACAAGGTA；
F13A1-15-2F:TGTAAAACGACGGCCAGTTTTTCCCAGGACTAGAGCACAA；
F13A1-15-2R:AACAGCTATGACCATGTACTGCGGAGGAAGGATGGT。
2. oligonucleotides as described in claim 1, which is characterized in that further include a pair of of sequencing primer, base sequence are as follows:
M13F:TGTAAAACGACGGCCAGT；
M13R:AACAGCTATGACCATG.
3. oligonucleotides as described in claim 1, which is characterized in that F13A1-1F:F13A1-1R, F13A1-2F:F13A1-
2R, F13A1-3F:F13A1-3R, F13A1-4F:F13A1-4R, F13A1-5F:F13A1-5R, F13A1-6F:F13A1-6R,
F13A1-7F:F13A1-7R, F13A1-8F:F13A1-8R, F13A1-9F:F13A1-9R, F13A1-10F:F13A1-10R,
F13A1-11F:F13A1-11R, F13A1-12F:F13A1-12R, F13A1-13F:F13A1-13R, F13A1-14F:F13A1-
The ratio of 14R, F13A1-15-1F:F13A1-15-1R and F13A1-15-2F:F13A1-15-2R are 1.
4. oligonucleotides as claimed in claim 2, which is characterized in that the ratio of M13F:M13R is 1.
5. application of the oligonucleotides in preparation detection coagulation factor deficiency kit as described in one of Claims 1 to 4.
6. a kind of method for detecting F13A1 gene mutation, comprising the following steps:
(1) genomic DNA in sample is extracted；
(2) it is expanded using the DNA at least pair for amplification primer pair (1) described in claim 1, obtains amplified production；
(3) it is sequenced using the amplified production in a pair of of sequencing primer pair (2) as claimed in claim 2, obtains the amplification
The base sequence of product；
(4) base sequence in (3) is compared with F13A1 gene wild-type reference sequence, determines whether mutational site deposits
?.
7. a kind of kit for detecting F13A1 gene mutation, the kit includes detection architecture pcr amplification reaction liquid, sequencing
System reaction solution, which is characterized in that the detection architecture pcr amplification reaction liquid includes at least a pair of of expansion described in claim 1
Increase primer, the sequencing system reaction solution includes a pair of of sequencing primer as claimed in claim 2.
8. kit as claimed in claim 7, which is characterized in that the detection architecture pcr amplification reaction liquid further includes 2 ×
PCR Buffer, dNTPs and KOD FX DNA Polymerase.
9. kit as claimed in claim 7, which is characterized in that the sequencing system reaction solution further includes EDTA, anhydrous second
Alcohol, 75% ethyl alcohol, HIDI and Bigdye Terminator V3.1.
10. kit as claimed in claim 7, which is characterized in that the sequencing system reaction solution further includes sequencing refined solution,
The sequencing refined solution includes exonuclease I and calf intestinal alkaline phosphatase.
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| CN113817816A (en) * | 2021-10-08 | 2021-12-21 | 济南艾迪康医学检验中心有限公司 | Primer, kit and method for detecting EPAS1 gene mutation |
| CN113913529A (en) * | 2021-12-01 | 2022-01-11 | 济南艾迪康医学检验中心有限公司 | Method, primer and kit for detecting c-195G > A site mutation of VHL gene |
| CN114480642A (en) * | 2022-01-05 | 2022-05-13 | 合肥艾迪康医学检验实验室有限公司 | Primer, method and kit for detecting SMAD4 gene full exon mutation |
| WO2022237888A1 (en) * | 2021-05-14 | 2022-11-17 | 医工瑞思(福建)工程研究中心有限公司 | Biomarker for detecting thrombus or blood coagulation related diseases and application thereof |
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| CN107841554A (en) * | 2017-11-24 | 2018-03-27 | 南京艾迪康医学检验所有限公司 | Detect the primer, kit and method of Hageman factor (F12) gene mutation |
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| CN111057758A (en) * | 2019-12-23 | 2020-04-24 | 福州艾迪康医学检验所有限公司 | Primer and method for detecting PSEN1 gene mutation and application thereof |
| CN112501279A (en) * | 2020-12-18 | 2021-03-16 | 长沙艾迪康医学检验实验室有限公司 | Primer, kit and method for detecting PIEZO1 gene mutation |
| WO2022237888A1 (en) * | 2021-05-14 | 2022-11-17 | 医工瑞思(福建)工程研究中心有限公司 | Biomarker for detecting thrombus or blood coagulation related diseases and application thereof |
| CN113817816A (en) * | 2021-10-08 | 2021-12-21 | 济南艾迪康医学检验中心有限公司 | Primer, kit and method for detecting EPAS1 gene mutation |
| CN113913529A (en) * | 2021-12-01 | 2022-01-11 | 济南艾迪康医学检验中心有限公司 | Method, primer and kit for detecting c-195G > A site mutation of VHL gene |
| CN114480642A (en) * | 2022-01-05 | 2022-05-13 | 合肥艾迪康医学检验实验室有限公司 | Primer, method and kit for detecting SMAD4 gene full exon mutation |
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