CN108660198A - A kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings - Google Patents
A kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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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- C12Q1/6869—Methods for sequencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention provides a kind of PCR SBT methods of CD36 antigens genotypings, it, which includes the following steps, prepares human gene group DNA；Amplimer is provided, expands the gene order of CD36 antigens respectively with polymerase chain reaction；Amplified production is subjected to double digestion purifying；Sequencing primer is provided, purified product is subjected to sequencing PCR reactions；Sequencing product is subjected to sodium acetate ethanol precipitation purifying, carries out Capillary Electrophoresis order-checking；The sequence of acquisition is analyzed by software, determines its genotype.The present invention also provides the reagents used in the above method.The present invention carries out sequence respectively by the gene order to the ends antigen 5` CD36 and 1 15 exons, obtains the oligonucleotide sequence of CD36 antigen gene partings, is accurately carried out shaping to its gene.The present invention has important practical significance for medical research unit, study of pharmacy and reagent exploitation unit.
Description
Technical field
The present invention relates to Genotyping detection methods, more particularly to one kind being used for human platelet's memebrane protein CD36 deletion forms
The molecular biology for detection of mutator, the invention further relates to the reagents that this method is applied.
Background technology
In recent years, due to the development for technology of transfusing blood, clinically Single-donor platelets infusion is own through becoming treatment decrease of platelet
One of the important means of.However as the increase of infusion number, it is defeated that blood platelet occur in some chronic, chronic infusion patients
Note invalid phenomenon.Therefore Inefficacy of Platelets Transfusion becomes clinically very stubborn problem, causes the extensive concern on defeated boundary.
The reason of Inefficacy of Platelets Transfusion, is divided into nonimmunologic factor and immune factor, in recent years, the CD36 antigenic deletions in immune factor
Caused infusion infectious-related complication becomes research hotspot.For this case, we research and develop sequencing kit detection CD36 antigens
The gene mutation point of missing, and speculated the relationship between CD36 antigenic deletions and Inefficacy of Platelets Transfusion.It is expected that CD36 antigens
Detection can become transfuse blood laboratory conventional detection project, to reduce the generation of platelet transfusion infectious-related complication.
People CD36 full length gene 32Kb are located at chromosome 7q.2, contain 15 exons.Exons 1,2 and 15 be non-volume
Code area, remaining 12 exon participate in coded amino acid, the N and C-terminal of exon 3 and 14 coding CD36 protein moleculars.
There are independent First Exons and 3 independent promoters on CD36 genes, without TATA boxes sequence and the islands CpG.To CD36
The research of gene mutation has lot of documents report.It has detected that a CD36 mutators more than 20 at present, causes generation CD36 anti-
Former deletion form.Gene mutation mechanism includes in mononucleotide substitution, base deletion, base insertion and mRNA montages processing
Exon skipping acts on, and the ripe mRNA that conversion generates is made to lack 1 or several exons.Gene mutation can generate two types
The CD36 antigenic deletions of type.The blood platelet and onthe surface of monocytes of 1 type CD36 missing individuals do not express CD36 antigens;2 types lack
It is that blood platelet does not express CD36 antigens to lose individual.II type ratio I types are more conventional.Blood platelet CD36 is lacked in white people very
Rare (0.3%), and (7%) frequency is very high in asian population (3-10%) and African crowd.It is had been reported that recently to German ethnic group
Blood donor carries out the choosing of CD36 (-) blood platelet hoof, does not up to the present find ' negative ' specimens also.In African crowd and the African U.S.
Lacked with CD36 in ethnic group related discovery gene mutation and asian population it is significantly different.CD36 genes have height more
State property.To African western part refuse disease hair area's individual CD36 genes carry out research be found that 24 mutant.
Epitope on blood platelet CD36 is referred to as Naka, and corresponding antibody is named as anti-Naka antibody.It is anti-
After repeatedly receiving HLA homotype platelet transfusions identifying in patient body for Inefficacy of Platelets Transfusion (PTR) occurs for Naka for the first time
Come.A large amount of anti-Naka antibody of Case Report and transfusion reaction were closely related later.Anti- Naka antibody can also cause immunity blood
Platelet reduces the diseases such as disease, early stage foetal death (repeat early fetal loss).F.Nakajima etc. (2008) exists
Anti- Naka is demonstrated in nearest research plays key during the occurrence and development of transfusion related acute lung injury (TRALI)
Effect.
Domestic at present to focus mostly in it research of CD36 with lipid metaboli and glycometabolism disease is associated with, shortage is to CD36
The system research and transfusion safety correlative study of whole gene polymorphism.It is lacked compared with other glycoprotein of blood platelet, CD36 is lacked
Losing has the higher characteristic of frequency, and CD36 lacks individual and usually do not show symptom, this undoubtedly increases CD36 deletion patients'
The generation of Adverse transfusion reaction.So far, oneself has found CD36 genetic mutations and metabolic syndrome, lactose intolerance, insulin
It resists, myocardial infarction, atherosclerosis, coronary heart disease, diabetes, hypertension, hyperlipidemia, angiogenesis, thrombotic diseases,
It is related to refuse a variety of diseases such as disease for apoplexy, Alzheimer disease.
The risk that caused may transfuse blood is lacked for blood platelet CD36, CD36 identification methods mainly have serological method at present
And methods of genotyping.Serological Identification G-Ag or the main granulocyte agglutination test of antibody method, granulocyte are immunized
Fluorescent test (GIFT), monoclonal antibody specificity G-Ag captive test (Monoclonal Antibody
Immobilization Of GranulocyteAntigen, MAIGA), the methods of flow cytometry and ELISA.Granulocyte is anti-
The methods of genotyping of original system mainly has PCR-RFLP, PCR-SSP, PCR-SSO etc..The experiment of Bux etc. shows gene point
Type method has same reliability with MAIGA methods to the parting of CD36, and GIFT has 15% parting fault rate.Current
The most important method of CD36 antigen gene partings is PCR-SSP (PCR- sequence specific primers), and this method needs to carry out multitube
Amplification, and PCR-SSP methods can only have distinctive specific position to be designed in design primer for certain, because
This can only carry out Genotyping to conventional CD36 antigens, some special new mutation sites are then difficult to clear.And CD36
PCR-SBT (PCR-Sequence Based Typing, the typing method of based on PCR sequencing) on classifying method can overcome
Defect and limitation are stated, is the method for most accurate parting.But CD36 antigen gene PCR-SBT classifying methods at this stage are also not
It is enough perfect, although also thering is laboratory to carry out Genotyping to CD36 antigens using the method for PCR-SBT, so far not to CD36 bases
Because same PCR conditions and dry PCR primer carry out system sequencing in PCR plate.Therefore, a kind of human platelet's memebrane protein is established
The PCR-SBT methods of CD36 deletion form mutators have great importance.
Invention content
Technical problem to be solved by the invention is to provide a kind of human platelet's memebrane protein CD36 deletion form mutators
PCR-SBT methods, to overcome the drawbacks described above in existing genotyping technique.For this purpose, the present invention uses following technical scheme
It to the gene order at the ends antigen 5` CD36 and 1-15 exons carry out respectively sequence gene carry out parting, including with
Lower step：
(1) amplimer is provided, primer drying is placed in detachable 96 hole PCR plate bottom using whizzer, is protected after sealer
Be stored in -20 DEG C it is spare；
(2) human gene group DNA is prepared；
(3) ends 5` of CD36 genes in human gene group DNA and the gene sequence of 1-15 exons are expanded respectively with polymerase chain reaction
Row；
(4) amplified production for obtaining step (3) carries out double digestion purifying；
(5) sequencing primer is provided, the purified product that step (4) obtains is subjected to sequencing PCR reactions；
(the 6 sequencing products for obtaining step (5) carry out sodium acetate-ethanol precipitation and purify, and carry out Capillary Electrophoresis order-checking;
(7) sequence that step (6) obtains is analyzed by software, determines its genotype.Purifying is required in the step (4) two
Kind enzyme is shrimp alkaline phosphotase and exonuclease I.
The present invention another the technical problem to be solved is that provide the above method used in reagent.For this purpose, the present invention adopts
With following technical scheme, it is made of the primer for amplification and the oligonucleotide sequencing primer for sequencing analysis；It is described to be used for
The primer of amplification is：
CD36-5'-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5'-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA
2 oligonucleotide sequencing primer sequences are as follows：
M13F:TGTAAAACGACGGCCAGT
M13R:CAGGAAACAGCTATGACC
In addition CD36-EXON1 increases by 2 sequencing primers：
Ploy-TF:GCTGTGTGGGGGATTTTTTTTTT
Ploy-TR:AGAGAAGAGAAAGCACTC。
Design of primers is the key that PCR amplification in the present invention, and method and software in relation to design of primers can be from internet
Upper free acquisition.Oligonucleolide primers designed by the present invention are according to being wrapped in mankind CD36 antigen gene sequences in GenBank
It includes the continuous oligonucleotide sequence including polymorphic site and designs acquisition.The amplification of the gene order of CD36 antigen systems is drawn
Object is NC_000007.13 (GI according to number in GenBank:224589819) sequence design.All forward direction amplimers
The ends 5` are connected with 16 base sequence TGTAAAACGACGGCCAGT in M13 carrier forward direction sequences, all reversed amplimers
5 ' ends are connected with 16 base sequence CAGGAAACAGCTATGACC on M13 carrier reverse sequences, by after connection it is all just
It is respectively provided with common joint sequence to amplimer, reversed amplimer, these joint sequences is then selected to draw as sequencing
Object.The present invention expands 16 genetic fragments of CD36 antigens with 16 pairs of Oligonucleolide primers respectively, it is ensured that CD36 antigen systems
Effective amplification of gene.The design of amplimer avoids the polymorphic site of CD36 antigen encoding sequences, avoids any prominent
The missing inspection of height.The design of sequencing primer can guarantee the clear sequence for accurately measuring institute's amplified fragments, by these sequences into
Row bidirectional sequencing, to which sample is carried out accurate genetic typing.The present invention passes through to the ends antigen 5` CD36 and 1-15 exons
Gene order carry out sequence respectively, obtain the oligonucleotide sequence of CD36 genes, accurately its gene carried out shaping.
With popularizing for DNA sequence analysis instrument, PCR-SBT technologies are widely used in clinical detection.All CD36 that high throughput obtains are anti-
The encoded sequence information of original system will in the application of genetic typing, genetic polymorphism detection, Investgation On Gene Frequencies of The Red analysis etc.
It is in widespread attention.Reagent provided by the present invention and method can be used as a kind of independent, widely used identification method, solve
The problem of gene order 16 segments of the ends CD365` and 1-15 exons obtain exact sequence plays PCR-SBT to CD36
High-throughput, the as a result accurate feature of genetic typing operation, the related application in the fields such as clinical blood transfusion medical research and science of heredity
It will be highly valued, have important practical significance for medical research unit, study of pharmacy and reagent exploitation unit.Especially
It is clear blood donation population CD36 antigen systems distribution situations, avoids being transfused the blood containing CD36 antibody, it is anti-to prevent granulocyte
The Adverse transfusion reaction that body generates, to improve the safety of blood.
Description of the drawings
Fig. 1 is detachable 96 hole of the invention PCR plate.Wherein 1 to 12 row, are often classified as 8 holes, two are classified as a person-portion, totally 16 hole.
PCR plate two is detachable between arranging.1st and 2 are classified as a person-portion, and 3,4 be another person-portion, and it is a plate so to analogize totally 6 person-portions.
Fig. 2 is detachable PCR plate schematic diagram, without cutting out, cutting.It is the PCR plate that primer is not added in figure.
Fig. 3 is the CD36 antigen gene PCR amplification electrophoresis patterns that sample is detected in the present invention.I is that CD36-5` expands piece
Section, 2 be CD36-EXON1 amplified fragments, and 3 be CD36-EXON2 amplified fragments, and 4 be CD36-EXON3 amplified fragments, and 5 be CD36-
EXON4 amplified fragments, 6 expand piece for CD36-EXON5, and 7 be CD36-EXON6 amplified fragments, and 8 expand piece for CD36-EXON7
Section, 9 be CD36-EXON8 amplified fragments, and 10 be CD36-EXON9 amplified fragments, and 11 be CD36-EXON10 amplified fragments, and 12 are
CD36-EXON11 amplified fragments, 13 be CD36-EXON12 amplified fragments, and 14 be CD36-EXON13 amplified fragments, and 15 be CD36-
EXON14,16 be CD36-EXON15 amplified fragments amplified fragments.
Fig. 4 is the part sequencing electrophoresis pattern that the present invention detects 3 sample CD36.A, G, C, T are respectively to be sequenced in figure
Four kinds of bases, A are adenine, and G is guanine, and C is cytimidine, and T is thymidine.
Specific embodiment mode
The content of present invention is described in further detail with reference to embodiments.
Embodiment I
This implementation specifically elaborates to the content of present invention so that blood donor carries out CD36 antigen gene partings as an example, institute of the present invention
A kind of PCR-SBT methods of the CD36 antigen genes parting used specifically include following steps
I, human gene group DNA, the PCR amplification template as subsequent step are prepared.Whole blood 400ml to be checked is taken, according to MagCore
HF16 automatic extracting instrument specifications extract genomic DNA and measured concentration..
2,16 pairs of amplimers and 4 sequencing primers are synthesized, particular sequence is shown in the sequence in foregoing summary, no longer superfluous
It states, amplimer is diluted to 10 μM with pure water;According to table 1 using whizzer primer, phenol red（100 μ g/ml) and it is pure
Water drying be placed in detachable 96 hole PCR plate bottom, be stored in after sealer -20 DEG C it is spare.
1 CD36 primers of table configure.
3, PCR Buffer are configured（360μl）：Prepare 10x buffer solutions (Lot:AC6901A, TaKaRa),dNTP (Lot
:BL3018, TaKaRa), ReadyPCR (lot:G9RP106, inno-train), pure water, prepared by system described in table 2, preserve
It is spare in -20 DEG C.
2 PCR Buffer configurations of table.
4, prepare r-Taq enzymes (Lot:AGY0124A, TaKaRa), expand template with the PCR prepared by step I
And prepared by step 3, PCR amplification system is prepared by system described in table 3, PCR prepared by step 2 is added in 25 μ l of every hole after mixing
In plate（Totally 16 holes/person-portion).
The PCR amplification system of 3 CD36 antigens of table.
400 μ l of total system in table 3 expand 95 DEG C of pre-degenerations 5min, DNA with PCR instrument (ABI, Vertiri) by following procedure
Double-strand is fully unlocked；95 DEG C are denaturalized 30s, 56 DEG C of annealing 30s, and primer is attached in template, and 72 DEG C of extension 1min extend required expansion
Increase segment, reacts 35 cycles；72 DEG C of 10min, amplified fragments fully extend.
5, the double digestion purifying of amplified production.Sample institute amplified fragments are detected, respectively take 5 μ lPCR products to carry out agarose solidifying
Gel electrophoresis, as shown in figure 3, determining the specificity of amplified fragments.2 μ l shrimp alkalinity are added in remaining PCR product takes out 10 μ l
The mixed liquor Exo-SAP of phosphatase (SAP) and exonuclease-I (Exo- I)（lot：160904, TBG) shrimp alkalinity, is utilized
Single-stranded specific Y-nucleic acid of the ends the nucleotide Y dephosphorylation function and exonuclease I (Exo- I) of phosphatase (SAP)
Excision enzyme function carries out amplified production purifying.2 μ l Exo-SAP, 37 DEG C of 30min enzymes are added in 10 μ l amplified production systems
Cut reaction, 80 DEG C of 15min enzymes inactivations.
6, sequencing PCR is carried out to PCR product.The dilution of 75ml pure water will be added in PCR product after being purified in step 3,
Mixing.Two sequencing primers described in invention content are diluted to a concentration of 3. 2 μm of ol/L with pure water, with
BigDyeterminator V3.1 sequencing Kit (American AB I companies) reagents prepare reaction system according to table 4,
Middle sequencing primer I is any one in M13F, M13R, Ploy-TF and Ploy-TR.Institute's test sample sheet is with the segment 1 of amplification purification:4
Template is used as after dilution, except CD36-EXON1 carries out M13F, M13R, Ploy-TF and Ploy-TR totally 4 reactions, Qi Tadou respectively
M13F, M13R totally 2 reactions are carried out respectively.It carries out expanding 96 DEG C of pre-degeneration by following procedure with PCR instrument (ABI, VerritiL)
2min, DNA double chain are fully unlocked；96 DEG C are denaturalized 10s, 50 DEG C of annealing 5s, and sequencing primer is attached on DNA profiling, 60 DEG C of extensions
4min, 29 cycles;72 DEG C extend amplified fragments 5min.
System is sequenced in the PCR of PCR product in 4 step 5 of table.
7, sequencing amplification PCR product is directly purified with sodium acetate/ethanol purification method.Amplification will be sequenced in step 4
PCR product is directly purified with sodium acetate/ethanol purification method.25 Μ Μ of I μ l EDTA I. are added directly in PCR product)
With 1 μ l sodium acetates (3 Μ)/absolute ethyl alcohol (25ml) mixed liquor, mixing, 2000g centrifuges 30min;Supernatant is removed, 80ml is added
70% ethyl alcohol, 2000g centrifuge 10min, remove supernatant, and 10 μ l formyl amine solvents are added after alcohol volatilization, and 95 DEG C are denaturalized 5min, fast
Speed cools down on ice.
8, the product prepared is carried out to 16 hole capillary high throughput electrophoresis sequencings, institute on ABI 3130XL sequenators
Sequencing result carries out sequence alignment using Seqman7.0 softwares, determines the genotype of CD36 antigen systems, as a result shows detection
The partial sequence of sample CD36 genes.Wherein Fig. 3 is the antigen gene PCR amplification electrophoresis pattern that sample is detected in the present invention.1
It is CD36-EXON1 amplified fragments for CD36-5` amplified fragments, 2,3 be CD36-EXON2 amplified fragments, and 4 expand for CD36-EXON3
Increase segment, 5 be CD36-EXON4 amplified fragments, and 6 expand piece for CD36-EXON5, and 7 be CD36-EXON6 amplified fragments, and 8 are
CD36-EXON7 amplified fragments, 9 be CD36-EXON8 amplified fragments, and 10 be CD36-EXON9 amplified fragments, and 11 be CD36-
EXON10 amplified fragments, 12 be CD36-EXON11 amplified fragments, and 13 be CD36-EXON12 amplified fragments, and 14 be CD36-
EXON13 amplified fragments, 15 be CD36-EXON14, and 16 be CD36-EXON15 amplified fragments amplified fragments.
Fig. 4 is the part sequencing electrophoresis pattern that the present invention detects 3 sample CD36 genes.A, G, C, T are respectively to survey in figure
Four kinds of bases of sequence, A are adenine, and G is guanine, and C is cytimidine, and T is thymidine.Sample 1 lacks for the 5th exons of CD36
Lose two base 329-330 del AC；Sample 2 is that one base A of the 12nd exons of CD36 is mutated into T；Sample 3 is CD36
The 13rd base 1228-1239 del of Exon deletion 12 ATTGTGCCTATT.
In conclusion reagent provided by the present invention and method can be used as a kind of independent, widely used identification method,
Solves the problems, such as the accurate Classification Identification of CD36 genes, performance PCR-SBT is accurate to CD36 genotypic results, high throughput is grasped
The characteristics of making, the related application in the fields such as clinical blood transfusion medical research and science of heredity will be highly valued, medicine ground
Study carefully unit, study of pharmacy and reagent exploitation unit to have great importance.Especially clear blood donation population G-Ag system
Distribution situation avoids the blood for being transfused the antibody containing granulocyte, to prevent the Adverse transfusion reaction of granulocyte antibody generation, will have
Effect prevents TRALI, to improve the safety of blood.
Sequence table
<110>Guangzhou Bleed Center
<120>A kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tgtaaaacga cggccagtaa aataagtttc gcaagctca 39
<210> 2
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
caggaaacag ctatgacctc ccccacacag cacattactg 40
<210> 3
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgtaaaacga cggccagtgc tgaatatctc agatatagg 39
<210> 4
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
caggaaacag ctatgaccgt atttatacag tagtgtcacc 40
<210> 5
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tgtaaaacga cggccagtcc tgtagtctat ccaaagtc 38
<210> 6
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
caggaaacag ctatgaccgt atggtaacag atgttttatt 40
<210> 7
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
tgtaaaacga cggccagtgt aggcattaga agcaagaa 38
<210> 8
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
caggaaacag ctatgaccag tcgcatcata tagagttg 38
<210> 9
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tgtaaaacga cggccagtaa agcgtcactc taaagc 36
<210> 10
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
caggaaacag ctatgaccat gacatttgcc aagtagaag 39
<210> 11
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
tgtaaaacga cggccagtct atctggcata ttctgtgt 38
<210> 12
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
caggaaacag ctatgaccaa gcatcttcct gtaatctg 38
<210> 13
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
tgtaaaacga cggccagtgg aatgtcgtct tcttgtg 37
<210> 14
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
caggaaacag ctatgaccaa ttatgccttg ccaatgc 37
<210> 15
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
tgtaaaacga cggccagtcc tcacctcaac atagtaaga 39
<210> 16
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
caggaaacag ctatgaccga gttaatacct agcagaacag 40
<210> 17
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
tgtaaaacga cggccagttg atctggctac ctaatggc 38
<210> 18
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
caggaaacag ctatgaccct ctgaatcatg cagtaaggg 39
<210> 19
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
tgtaaaacga cggccagtat ggactacact ggaggag 37
<210> 20
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
caggaaacag ctatgacctt ggaagatgca gaagaaca 38
<210> 21
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
tgtaaaacga cggccagttt catgcttggc tattgagtt 39
<210> 22
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
caggaaacag ctatgacctc tttcttctgc cctaat 36
<210> 23
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
tgtaaaacga cggccagtgc ctgaaagctt tacatattg 39
<210> 24
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
caggaaacag ctatgacccc ataggaagaa atcgacc 37
<210> 25
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
tgtaaaacga cggccagtaa ccttgacatt cgattgg 37
<210> 26
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
caggaaacag ctatgaccga gatgctatca aatgctca 38
<210> 27
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
tgtaaaacga cggccagtta tttcagttcc ccgaga 36
<210> 28
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
caggaaacag ctatgacctt tgttcaattg gatcat 36
<210> 29
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
tgtaaaacga cggccagtct gatgactaac accaatagag 40
<210> 30
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
caggaaacag ctatgacctg gacaactttg gcacaa 36
<210> 31
<211> 35
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
tgtaaaacga cggccagtca tcatttccac aactg 35
<210> 32
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
caggaaacag ctatgaccat tagcctagaa caaagtggta 40
<210> 33
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
tgtaaaacga cggccagt 18
<210> 34
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
caggaaacag ctatgacc 18
<210> 35
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
gctgtgtggg ggattttttt ttt 23
<210> 36
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
agagaagaga aagcactc 18
Claims (5)
1. a kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings, it is characterised in that it is by being used for
The primer of amplification and for sequencing analysis oligonucleotide sequencing primer form；The primer for amplification is CD36 antigens
The amplimer of gene order, method include the following steps：
(1) amplimer is provided, primer drying is placed in detachable 96 hole PCR plate bottom using whizzer, is protected after sealer
Be stored in -20 DEG C it is spare；
(2) human gene group DNA is prepared；
(3) gene order of the ends CD36 antigens 5` and 1-15 exons in human gene group DNA is expanded respectively with polymerase chain reaction；
(4) amplified production for obtaining step (3) carries out double digestion purifying；
(5) sequencing primer is provided, the purified product that step (4) obtains is subjected to sequencing PCR reactions；
(6) the sequencing product for obtaining step (5) carries out sodium acetate-ethanol precipitation purifying, carries out Capillary Electrophoresis order-checking；
(6) sequence that step (6) obtains is analyzed by software, determines its genotype.
2. the PCR-SBT methods and reagent of a kind of platelet membrane proteins CD36 antigen gene partings according to claim I,
It is characterized in that the amplimer in the step (2) is expanded for 16 pairs of oligonucleotides and is shown outside the ends antigen 5` CD36 and 1-15 respectively
The gene order of the gene order of son
The amplimer of the gene order of CD36 antigen systems
CD36-5’-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5’-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA。
3. the PCR-SBT methods and reagent of a kind of platelet membrane proteins CD36 antigen gene partings according to claim 1,
It is characterized in that the sequencing primer in the step (5) is that 2 oligonucleotide sequencing primer sequences are as follows：
M13F：TGTAAAACGACGGCCAGT,
M13R：CAGGAAACAGCTATGACC；
In addition CD36-EXON1 increases by 2 sequencing primers：
Ploy-TF:GCTGTGTGGGGGATTTTTTTTTT
Ploy-TR:AGAGAAGAGAAAGCACTC。
4. a kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings as described in claim 1,
It is characterized in that two kinds of enzymes in the step (3) needed for purifying are shrimp alkaline phosphotase and exonuclease I.
5. a kind of the PCR-SBT methods and reagent of platelet membrane proteins CD36 antigen gene partings, it is characterised in that it is by being used for
The primer of amplification and for sequencing analysis oligonucleotide sequencing primer form；The primer for amplification is CD36 antigens
The amplimer of the gene order of system,
CD36-5'-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5'-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA
2 oligonucleotide sequencing primer sequences are as follows
M13F : TGTAAAACGACGGCCAGT
M13R : CAGGAAACAGCTATGACC。
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