CN106399552A - ARMS primer for detecting CYP2C9 gene polymorphism - Google Patents
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Abstract
The invention discloses an ARMS primer for detecting CYP2C9 gene polymorphism. Aiming at sequence changes in every SNP of CYPC2C9*2(rs1799853, C430T, Arg144Cys) and CYP2C9*3(rs1057910, A1075C, Ile359Leu), the specificity ARMS primer and shared primers are designed. Mismatched base group (base group is marked with underline) is introduced to the second or third place of the 3' terminal end of the specificity ARMS primer so as to enlarge efficiency gap generating when specificity ARMS primer 3' terminal base are complementary and are not complementary to the SNP locus base on the DNA template, thus sharply improving specificity and accuracy of the detection.
Description
Technical field
The invention belongs to life sciences and biological technical field, dash forward particularly to detection CYP2C9 gene the 3rd, 7 exons
The ARMS primer becoming, using regular-PCR technology and gel electrophoresiss, can be used for the mutation in quick detection CYP2C9 gene polymorphic site
Situation.
Background technology
Human gene's polymorphism is illustrating human body to disease, the susceptibility of poisonous substance and toleration, to Disease Clinical performance
Multiformity, and all play an important role in the reactivity of Drug therapy.
CYP2C9 gene is located on No. 10 chromosome of the mankind, overall length 83529bp, totally 10 exons.CYP2C9 is thin
Important member in born of the same parents' cytochrome p 450 enzyme (CYP) the second subfamily, accounts for the 20% of hepatomicrosome P450 Tot Prot.CYP2C9 joins
Multiple with anticoagulant, anticonvulsant, antidiabetic drug, nonsteroidal antipyretic-antalgic anti-inflammatory agent, antihypertensive and diuretic etc.
The hydroxylation metabolism of medicine, wherein warfarin, tolbutamide and 5,5-Diphenyl-2,4-imidazolidinedione are the narrower medicine of therapeutic index.CYP2C9 activity
Change may result in these medicine bulk concentrations large change, even results in the generation of severe drug untoward reaction.
CYPC2C9*2 (rs1799853, C430T, Arg144Cys) and CYP2C9*3 (rs1057910, A1075C, Ile359Leu) is all
CYP2C9 enzymatic activity is led to reduce, CYP2C9*3 homozygotic individual enzymatic activity is only this site wild-type homozygote genotype individuals
The 4-6% of (carrying CYP2C9*1 or Arg144/Ile359 allele).In Chinese population, the frequency of CYPC2C9*2 is 0%,
The frequency of CYPC2C9*3 is 3%.CYP2C9 genetic polymorphism leads to its Enzyme activities, thus lead to drug metabolism race and
Individual variation phenomenon.
The method that can be used for CYP2C9 gene type is a lot, including such as DNA direct sequencing, hybridization hybrid chip method, pyrophosphoric acid
Sequencing, denaturing high-performance liquid chromatography, real time fluorescent quantitative method, restriction fragment length polymorphism (RFLP) and amplification resistance
Hinder abruptly-changing system (ARMS) etc..Wherein, DNA sequencing method is the goldstandard of gene type, but the method is deposited in clinical practice
In some restrictions：The sensitivity of detection is not high, time-consuming, complex operation, easily pollutes, and flux is little etc..Restriction fragment length polymorphism
Property is only applicable to the situation that there is suitable specific restriction endonuclease recognition sequence near mutational site, and application limitation is relatively
Greatly.There is false positive rate height in hybridization hybrid chip detection method, annealing temperature requires accurately, the not high problem of specificity；Taqman method
Probe preparation and matching used instrument and equipment are expensive, and accuracy is not high.
Amplification refractory mutation system does not have 3 ' → 5 ' 5 prime excision enzyme activity using Taq enzyme, when 3 ' terminal bases occur mispairing,
Can not effective amplification principle.
Gene type detection generally includes two complementary PCR reactions, common using identical DNA profiling and an identical
There are primer and reaction condition, differ only in, wild type ARMS primer 3 ' end different from the ARMS primer of total primer pairing
Mate with wild-type template, by mutational site design at 3 ' ends during saltant type ARMS design of primers, with saltant type template matching, make
Two reaction selectivity expand specific DNA profiling, and that is, the extension of 3 ' ends mismatch primers is hindered, generally also at 3 ' ends
The 2-4 base in end artificially arranges base mismatch, to improve the selection amplification property of mismatched primers.
Content of the invention
The purpose of the present invention is to provide one kind to be based on for the problems of existing detection CYP2C9 gene pleiomorphism
The method of ARMS-PCR detects the ARMS primer of CYP2C9 gene pleiomorphism, can be used for quick, effective detection person under test CYP2C9
The catastrophe in gene polymorphic site.
In order to realize foregoing invention purpose, the technical solution adopted in the present invention is as follows：
For CYPC2C9*2 (rs1799853, C430T, Arg144Cys) and CYP2C9*3 (rs1057910, A1075C,
Ile359Leu) each SNP sequence change and design specificity ARMS primer and general primer, specificity ARMS primer in
3 ' end seconds or three introduce base mismatch (underscore labelling base), with this expand specific primer 3 ' terminal bases with
SNP site base complementrity and the not gap between amplification efficiency under complementary case on DNA masterplate, thus be greatly improved the special of detection
Property and accuracy.Wherein：
A kind of ARMS primer of detection CYP2C9 gene pleiomorphism, including：
Detect the primer of CYPC2C9*2 (rs1799853, C430T, Arg144Cys) gene, its base sequence is：
General primer 2C9*2-CR：CCAGTAAGGTCAGTGATATGGAGTAG；
For detection site CYPC2C9rs1799853, the specific primer of C is one of following nucleotide sequences：
2C9*2-W1：GGGAAGAGGAGCATTGAGGGCC；
2C9*2-W2：GGGAAGAGGAGCATTGAGGTCC；
For detection site CYPC2C9rs1799853, the specific primer of T is one of following nucleotide sequences：
2C9*2-M1：GGGAAGAGGAGCATTGAGGGCT；
2C9*2-M2：GGGAAGAGGAGCATTGAGGTCT；
Detect the primer of CYPC2C9*3 (rs1057910, A1075C, Ile359Leu) gene, its base sequence is：
General primer 2C9*3-CR：ACCTTGGGAATGAGATAGTTTCTGAAT；
For detection site CYPC2C9rs1057910, the specific primer of A is one of following nucleotide sequences：
2C9*3-W1：GTGCACGAGGTCCAGAGATAGA；
2C9*3-W2：GTGCACGAGGTCCAGAGATAAA；
For detection site CYPC2C9rs1057910, the specific primer of C is one of following nucleotide sequences：
2C9*3-W1：GTGCACGAGGTCCAGAGATAGC；
2C9*3-W2：GTGCACGAGGTCCAGAGATAAC.
In a preferred embodiment of the invention, when detection site is for CYPC2C9*2 (rs1799853), using drawing
Thing is combined as sharing downstream primer 2C9*2-CR and ARMS primer pair 2C9*2-W2/2C9*2-M2.
In a preferred embodiment of the invention, when detection site is for CYPC2C9*3 (rs1057910), using drawing
Thing is combined as sharing downstream primer 2C9*3-CR and ARMS primer pair 2C9*3-W2/2C9*3-M2.
The present invention is simple to operate, testing result accurately and reliably, specifically have the beneficial effect that：
1st, avoided Sequences similar region between family gene, it is to avoid the non-spy that between family gene, the proximate region of sequence causes
Specific amplification, the design of this primer sets has the advantages that high specificity.
2nd, the base mispairing introducing, improves the identification ability of primer pair SNP, enables primer effectively to identify a base
Difference, expand the most suitable annealing region, it is to avoid because the false positive that annealing temperature requires accurately led to testing result is sent out
Raw, improve detection accuracy and reliability.
3rd, detection method is simple, without expensive device it is only necessary to PCR and electrophoresis equipment just can complete to test.
4th, operate rapid and convenient, the detection used time is short, typing work can be completed in 2 hours.
Brief description
Fig. 1 is the typing electrophoretogram of CYPC2C9*2 (rs1799853) and CYPC2C9*3 (rs1057910).Used by figure
Marker be Takara company 50bp marker.Nethermost band is 50bp, and second from the bottom is 100bp.Left side
Be detection sample CYP2C9*2, expand from left to right use primer be respectively 2C9*2-W1,2C9*2-M1,2C9*2-W2 and
2C9*2-M2；Right side is detection sample CYP2C9*3, and the primer expanding use from left to right is respectively 2C9*3-W1,2C9*3-
M1,2C9*3-W2 and 2C9*3-M2.
Fig. 2 is the Sanger sequence verification collection of illustrative plates of CYPC2C9*2.In figure uses Sanger sequencing detection to sample
CYPC2C9*2 (rs1799853, C430T, Arg144Cys), in figure frame is shown that pleomorphism site.
Fig. 3 is the Sanger sequence verification collection of illustrative plates of CYPC2C9*3.Sample is used with Sanger sequencing detection CYPC2C9*3
(rs1057910, A1075C, Ile359Leu), in figure frame is shown that pleomorphism site.
Specific embodiment
According to ARMS-PCR principle, preferably 8 specific primers of CYPC2C9, including for rs1799853 for the present invention
Four specific primers (2C9*2-W1/2C9*2-M1,2C9*2-W2/2C9*2-M2)；Four for rs1057910 special
Property primer (2C9*3-W1/2C9*3-M1,2C9*3-W2/2C9*3-M2).This 8 specific primers 3 ' end bases with corresponding
Two kinds of different bases of SNP site are mutually complementary, and the second in 3 ' ends or the 3rd introducing base mismatch, base mismatch
Introducing can expand the amplification efficiency difference after specific primer is combined with complementary masterplate and incomplementarity masterplate, make specificity
The optimal annealing range of primer expands, it is to avoid the generation of non-specific amplification, improves accuracy and the specificity of detection.Through reality
Test tries, and these primers can be annealed in the range of 55 degree～60 degree, carries out specific amplification, maximum possible avoid vacation
Positive generation.
The present invention further preferably 2 shared downstream primers, including the 2C9*2-CR for rs1799853；For
The 2C9*3-CR of rs1057910.This 2 primers pass through to detect in human genome database, have avoided base between family gene
Consistent region because of sequence, it is to avoid the combination with the outer template sequence of purpose fragment, improves the specificity of amplification.
The present invention can choose common for any one in the specific primer of same detection site and its corresponding downstream
Combined with primer, form the detection primer combination that this is point, realize the specific amplification detection to detection site.Detection site is
During CYPC2C9*2 (rs1799853), it is combined as sharing downstream primer 2C9*2-CR using primer, and ARMS primer pair 2C9*
2-W2/2C9*2-M2,2C9*2-W2/2C9*2-M2 two is to any pair in specific primer；Inspection detection site is CYPC2C9*3
(rs1057910), when, it is combined as sharing downstream primer 2C9*3-CR using primer, and ARMS primer pair 2C9*3-W2/2C9*
3-M2,2C9*3-W2/2C9*3-M2 two is to any pair in specific primer.
With reference to specific embodiments and the drawings, the present invention is expanded on further.
A kind of CYPC2C9 gene type detection based on ARMs-PCR method of embodiment 1
Pcr amplification reaction liquid amplification reaction solution is 20ul, using the Q5hifi DNA pilymerase reagent of NEB company
Dress, comprises 5xQ5buffer, 5xGC buffer, dNTP, Q5hifi DNA pilymerase and 5xGC enhancer.PCR is anti-
Every person-portion 2 should be managed, manage as specific reaction pipe for 1～No. 2.Specifically it is shown in Table 1
Table 1
Embodiment 2
A kind of operating procedure of the CYPC2C9 gene type detection based on ARMs-PCR method
(1) extract person under inspection's whole blood DNA or saliva DNA, be diluted to the DNA solution of 20ng/ul；
(2) PCR amplification：Detection is carried out on Standard PCR instrument, and reaction condition is shown in Table 2：
Table 2
PCR amplification system preparation of reagents method is shown in Table 3：
Table 3
| 5x Q5buffer | 4ul |
| 5x GC buffer | 4ul |
| 10mM dNTP | 0.4ul |
| Q5hifi DNA pilymerase | 0.2ul |
| 5xGC enhancer | 4ul |
| template DNA | 20ng |
| 10uM Forward Primer | 1ul |
| 10uM Reverse Primer | 1ul |
| Nuclease-free water | up to 20ul |
(3) electrophoresis：2% agarose gel electrophoresiies, 110V, 25min.Gel imaging system is taken pictures, obtains electrophoresis knot
Fruit is schemed.According to the presence or absence of purpose band, carry out gene type assay.
If in PCR reaction tube, such as no spawn band, only primer dimer band (less than 100bp), it is judged as that amplification is lost
Lose.If in PCR reaction tube, wild primers are augmented with purpose product, and do not have band after Idiotype ARMS primer amplification, then sentence
Read as wild type；If in PCR reaction tube, Idiotype ARMS primer amplification does not have purpose product, and has after wild primers amplification
Band, then interpretation is mutant homozygous type；If in PCR reaction tube, wild primers and Idiotype ARMS primer amplification are all purposeful
Product, then interpretation is heterozygous.
Marker used by figure is the marker of the 50bp of Takara company.Sample is the genomic DNA of same person.
Left side be detection sample CYP2C9*2, expand from left to right use primer be respectively 2C9*2-W1,2C9*2-M1,
2C9*2-W2 and 2C9*2-M2.Can two pairs of primers be all that wild primers are augmented with purpose product as seen from the figure, and special
There is no band, interpretation is wild type after type ARMS primer amplification；
Right side be detection sample CYP2C9*3, expand from left to right use primer be respectively 2C9*3-W1,2C9*3-M1,
2C9*3-W2 and 2C9*3-M2.Can two pairs of primers be all that wild primers are augmented with purpose product as seen from the figure, and special
There is no band, interpretation is wild type after type ARMS primer amplification.
Through the checking of DNA direct Sequencing, as shown in Figure 2 and Figure 3, completely consistent with the above results, the CYP2C9*2 of sample with
CYP2C9*3 is wild type.
The above results show, the ARMS primer of the present invention is reliable come the method to detect CYP2C9 gene pleiomorphism, sensitivity
Height, simple to operate, sentence read result is objective.
Sequence table
SEQUENCE LISTING
<110>Upper Shanghai's style Sen Nuo biotech inc
<120>For detecting the ARMS primer of CYP2C9 gene pleiomorphism
<130>
<160> 10
<170> Patent In version 3.3
<210> 1
<211> 26
<212> DNA
<213>Artificial sequence
<400> 1
ccagtaaggt cagtgatatg gagtag 26
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
gggaagagga gcattgaggg cc 22
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
gggaagagga gcattgaggt cc 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
gggaagagga gcattgaggg ct 22
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
gggaagagga gcattgaggt ct 22
<210> 6
<211> 27
<212> DNA
<213>Artificial sequence
<400> 6
accttgggaa tgagatagtt tctgaat 27
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
gtgcacgagg tccagagata ga 22
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
gtgcacgagg tccagagata aa 22
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence
<400> 9
gtgcacgagg tccagagata gc 22
<210> 10
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
gtgcacgagg tccagagata ac 22
Claims (3)
1. a kind of ARMS primer of detection CYP2C9 gene pleiomorphism is it is characterised in that include：
Detect the primer of CYPC2C9*2 (rs1799853, C430T, Arg144Cys) gene, its base sequence is：
General primer 2C9*2-CR：CCAGTAAGGTCAGTGATATGGAGTAG；
For detection site CYPC2C9rs1799853, the specific primer of C is one of following nucleotide sequences：
2C9*2-W1：GGGAAGAGGAGCATTGAGGGCC；
2C9*2-W2：GGGAAGAGGAGCATTGAGGTCC；
For detection site CYPC2C9rs1799853, the specific primer of T is one of following nucleotide sequences：
2C9*2-M1：GGGAAGAGGAGCATTGAGGGCT；
2C9*2-M2：GGGAAGAGGAGCATTGAGGTCT；
Detect the primer of CYPC2C9*3 (rs1057910, A1075C, Ile359Leu) gene, its base sequence is：
General primer 2C9*3-CR：ACCTTGGGAATGAGATAGTTTCTGAAT；
For detection site CYPC2C9rs1057910, the specific primer of A is one of following nucleotide sequences：
2C9*3-W1：GTGCACGAGGTCCAGAGATAGA；
2C9*3-W2：GTGCACGAGGTCCAGAGATAAA；
For detection site CYPC2C9rs1057910, the specific primer of C is one of following nucleotide sequences：
2C9*3-W1：GTGCACGAGGTCCAGAGATAGC；
2C9*3-W2：GTGCACGAGGTCCAGAGATAAC.
2. as claimed in claim 1 a kind of ARMS primer of detection CYP2C9 gene pleiomorphism it is characterised in that working as detecting position
When point is for CYPC2C9*2 (rs1799853), it is combined as sharing downstream primer 2C9*2-CR and ARMS primer pair using primer
2C9*2-W2/2C9*2-M2.
3. as claimed in claim 1 a kind of ARMS primer of detection CYP2C9 gene pleiomorphism it is characterised in that working as detecting position
When point is for CYPC2C9*3 (rs1057910), it is combined as sharing downstream primer 2C9*3-CR and ARMS primer pair using primer
2C9*3-W2/2C9*3-M2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996244B (en) * | 2020-10-28 | 2022-05-06 | 浙江绍兴鼎晶生物医药科技股份有限公司 | Composition for detecting single nucleotide polymorphism and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789642A (en) * | 2014-01-20 | 2015-07-22 | 北京乐普医疗科技有限责任公司 | Primer for detecting polymorphism of mononucleotide, and kit and detection method thereof |
-
2016
- 2016-11-09 CN CN201610985360.0A patent/CN106399552A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789642A (en) * | 2014-01-20 | 2015-07-22 | 北京乐普医疗科技有限责任公司 | Primer for detecting polymorphism of mononucleotide, and kit and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HIRATSUKA M等: "High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5" nuclease chain reaction assay", 《BIOL PHARM BULL.》 * |
| ZHIYU CHEN等: "A study of correlation between CYP2C9 gene polymorphism and Warfarin maintenance dose in anticoagulant therapy among Han people in Yunnan of China", 《J.MED.GENET.GENOMICS》 * |
| 黄璐琦: "《分子生药学》", 30 September 2006, 北京：北京大学医学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996244B (en) * | 2020-10-28 | 2022-05-06 | 浙江绍兴鼎晶生物医药科技股份有限公司 | Composition for detecting single nucleotide polymorphism and application thereof |
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