CN108018357A - AGTR1 gene detecting kits and detection method - Google Patents
AGTR1 gene detecting kits and detection method Download PDFInfo
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- CN108018357A CN108018357A CN201710154081.4A CN201710154081A CN108018357A CN 108018357 A CN108018357 A CN 108018357A CN 201710154081 A CN201710154081 A CN 201710154081A CN 108018357 A CN108018357 A CN 108018357A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention relates to genetic engineering and biology field, a kind of AGTR1 gene detecting kits, including for carrying out the primer sets of specific amplification to the nucleic acid fragment in the site containing rs5186, the primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:4, and anti-sense primer SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8.Present invention also offers a kind of gene tester.The kit of the present invention expands the nucleic acid fragment containing above-mentioned rs5186 sites by using specificity amplification primer group so that low to detection sensitivity height, the false positive rate of AGTR1 gene mutations；The kit of the present invention is detected using second generation high throughput gene sequencing technology so that detection cycle is short, and flux is high.
Description
Technical field
The present invention relates to genetic engineering and biology field, is tried more specifically to a kind of AGTR1 genetic tests
Agent box and detection method.
Background technology
Hypertension is disease of the inherent cause caused by with environmental factor interaction.Research shows, renin-angiotensin
Prime system is united（RAS）Play an important roll in terms of adjusting blood pressure and maintaining internal water, electrolyte balance, take part in the high blood of primary
The occurrence and development of pressure.The main physiological activity substance angiotensin II of this system（Angiotensin II, AT II）In the heart
Play an important role in the occurrence and development of flesh plumpness, it mainly passes through its I receptor（angiotensin Ⅱ type1
Receptor, AGTR1）Mediation so as to playing its effect.
AGTR1 genes are located at human chromosome 3q21-25, its coded product mainly mediates the Cardiovascular of AT II.Quan Ji
Because most important candidate's tumor susceptibility gene that AGTR1 genes are EH morbidities, the generation of AGTR1 gene pleiomorphisms and EH are studied in group scanning
Development is related.More and more researchs show that angiotensin Ⅱ receptor type Ⅰ (AGTR1) gene pleiomorphism is to high blood in recent years
Pressure, kidney trouble, vascular diseases, coronary heart disease, myocardial hypertrophy, the occurrence and development important of heart failure and diabetes.
AGTR1 genetic mutations not only can provide reference information for the diagnosis of the examination of these disease vulnerable crowds, prevention and disease, more have
Prestige can provide theoretical and case selection foundation for the clinical practice of AGTR1 antagonists, and can be having for the critical illnesses such as myocardial infarction
Correlation gene treatment provides theoretical and experiment basis.By being detected to the AGTR1 genes of patient, the gene of specific site is obtained
Type, the method using genetic test result as the information of intermediate result, is not belonging to the diagnostic method of disease；With the detection of genotype
As a result the selection of medicine is determined, it is possible to realize personalized medicine, avoid to immunodepressant adverse reaction.
Currently used AGTR1 detection method of gene mutation mainly uses gene chips.But gene chips sensitivity
It is low, false positive rate is high, time-consuming longer, flux is small.
Therefore, it is necessary to a kind of kit and detection method of new detection AGTR1 genes mutation so as to AGTR1
The high sensitivity of gene mutation, false positive rate is high, and detection cycle is short.
The content of the invention
It is an object of the invention to provide a kind of AGTR1 gene detecting kits and detection method, it is intended to solves existing skill
AGTR1 genetic tests sensitivity is low in art, false positive rate is high, cycle length, the small technical problem of flux.
A kind of AGTR1 gene detecting kits, including for carrying out specific expansion to the nucleic acid fragment in the site containing rs5186
The primer sets of increasing, the primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID
NO:4, and anti-sense primer SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8.
Preferably, the gene detecting kit further includes connector, and the connector includes connector A, connector B, connector C, connects
At least one of head D；The connector A is by SEQ ID NO:9 and SEQ ID NO:10 compositions；The connector B is by SEQ ID
NO:11 and SEQ ID NO:12 compositions；The connector C is by SEQ ID NO:13 and SEQ ID NO:14 compositions；The connector D by
SEQ ID NO:15 and SEQ ID NO:16 compositions.
Preferably, the gene detecting kit further includes sequencing primer SEQ ID NO:17, the sequencing primer is used for
The library molecule in the site containing rs5186 is sequenced.
Preferably, phosphorylation modification is passed through in 5 ' ends of the sequencing primer.
A kind of gene tester, comprises the following steps：
A, using amplimer group, the sample in the site containing rs5186 is expanded, obtains amplified production；It is it is characterized in that, described
Primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:4, and downstream draws
Thing SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in rs5186 sites
Type.
Preferably, the step A comprises the following steps：
A1, using the first primer sets expand AGTR1 genetic fragments, obtain the first amplified production；First primer sets include first
Sense primer SEQ ID NO:1 or SEQ ID NO:2, and the first anti-sense primer SEQ ID NO:5 or SEQ ID NO:6；；
A2, using the first amplified production as template, using the second primer sets amplification the site containing rs5186 nucleic acid fragment, obtain the second expansion
Increase production thing；Second primer sets include the second sense primer SEQ ID NO:3 or SEQ ID NO:4, and the second downstream draws
Thing SEQ ID NO:7 or SEQ ID NO:8.
Preferably, further included in the step B and the step of USER enzymes are cut is added into the second amplified production, it is described
Second amplified production forms the first cohesive end through USER cleavages.
Preferably, the connector includes at least one of connector A, connector B, connector C, connector D；The connector A is by SEQ
ID NO:9 and SEQ ID NO:10 compositions；The connector B is by SEQ ID NO:11 and SEQ ID NO:12 compositions；The connector C
By SEQ ID NO:13 and SEQ ID NO:14 compositions；The connector D is by SEQ ID NO:15 and SEQ ID NO:16 compositions.
Preferably, the sequencing primer for being used to the library molecule in the site containing rs5186 be sequenced in step C is SEQ ID
NO:17。
Preferably, phosphorylation modification is passed through in 5 ' ends of the sequencing primer.
AGTR1 gene detecting kits provided by the invention, using specific PCR amplimer group to site containing rs5186
Fragment expanded, reduce the possibility for expanding multiple target sites, improve to rs5186 sites amplification specificity and
Sensitivity, reduces false positive rate；Meanwhile using second generation high throughput gene sequencing technology so that the inspection to rs5186 sites
The survey cycle is short, and flux is high.
Brief description of the drawings
Fig. 1 is the PAGE glue electrophoretogram of 3rd embodiment Chinese library molecule.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right
The present invention is further elaborated.
Present invention proposition first embodiment, a kind of AGTR1 gene detecting kits, including for the site containing rs5186
Nucleic acid fragment carries out the primer sets of specific amplification, and the primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、
SEQ ID NO:3 or SEQ ID NO:4, and anti-sense primer SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ
ID NO:8。
The AGTR1 gene detecting kits that this programme provides, including specificity amplification primer group, the specificity amplification primer
Group is capable of the nucleic acid fragment in specific amplification site containing rs5186, reduces the possibility for expanding non-purpose fragment, so that
It is low to the high sensitivity of rs5186 site primers, false positive rate.
Preferably, the kit further includes connector, and the connector is included in connector A, connector B, connector C, connector D extremely
Few one kind；The connector A is by SEQ ID NO:9 and SEQ ID NO:10 compositions；The connector B is by SEQ ID NO:11 and SEQ
ID NO:12 compositions；The connector C is by SEQ ID NO:13 and SEQ ID NO:14 compositions；The connector D is by SEQ ID NO:
15 and SEQ ID NO:16 compositions.Wherein, sequence label AATT is contained on the connector A；Contain sequence label on the connector B
TTAA；Contain sequence label CCGG on the connector C；Contain sequence label GGCC on the connector D.
It should be noted that when kit using the present invention is in same system while to the rs5186 of different samples
When site is detected, since the sequencing primer used in sequencing procedure is identical, it is therefore desirable to use and contain different labels
The connector of sequence connects different samples respectively, by the way that sequence label is sequenced, is without carrying out repeatedly sequencing to sample
The sequencing result of different samples can effectively be distinguished.The joint sequence of this programme design, will not since 5 ' ends are free of phosphate group
Occur from connecting, so as to improve the joint efficiency between connector and amplified production；And the four kinds of label sequences combined in joint sequence
Base identification is simple during being listed in subsequent detection, easily distinguishable, and will not produce secondary structure；Sequence label length is 4bp,
Reduce design and detection difficulty.
Preferably, the SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15 5 ' ends
On contain biotin labeling, and biotin labeling is fixed on the magnetic bead containing Streptavidin or Avidin in advance.Utilize this
Biotin labeling, very easily can isolate the nucleic acid fragment of site containing rs5186 and connector after coupled reaction
Come.
Preferably, the kit further includes sequencing primer, and the sequencing primer includes being used to detect rs5186 sites
Sequencing primer SEQ ID NO:17.The sequencing primer of this programme design, is connected to rs5186 location proximates area in sequencing procedure
Domain so that in sequencing procedure, it is only necessary to which the sequencing of fewer number can determine that the genotype in rs5186 sites.
Preferably, the phosphorylated modification in 5 ' ends of the sequencing primer.The sequencing primer of this programme is suitable for using company
Connect PCR sequencing PCR to be detected rs5186 sites, this programme causes the 5 ' ends that probe is attached to sequencing primer are sequenced.
The present invention proposes second embodiment, and a kind of gene tester, comprises the following steps：
A, using amplimer group, the sample in the site containing rs5186 is expanded, obtains amplified production；It is it is characterized in that, described
Primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:4, and downstream draws
Thing SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in rs5186 sites
Type.
Gene tester provided by the invention, using the nucleic acid piece in specificity amplification primer group amplification site containing rs5186
Section, improves the sensitivity to rs5186 site primers, reduces the false positive rate of detection.
Preferably, the step A comprises the following steps：
A1, using the first primer sets amplification AGTR1 genes No. 10 allele regions, obtain the first amplified production；Described first draws
Thing group includes the first sense primer SEQ ID NO:1 or SEQ ID NO:2, and the first anti-sense primer SEQ ID NO:5 or SEQ
ID NO:6；；
A2, using the one 3 amplified production as template, using the second primer sets amplification the site containing rs5186 nucleic acid fragment, obtain second
Amplified production；Second primer sets include the second sense primer SEQ ID NO:3 or SEQ ID NO:4, and the second downstream
Primer SEQ ID NO:7 or SEQ ID NO:8.
This programme is special to the nucleic acid fragment progress two-wheeled in the site containing rs5186 by designing two pairs of PCR amplification primer sets
Property amplification, can effectively ensure in the second amplified production the not non-specific amplification caused by primer pairing specificity is not strong
Pollution, reduces the possibility for expanding multiple target sites, adds specificity and the sensitivity of amplification.
Preferably, further included in the step B and the step of USER enzymes are cut is added into the second amplified production, it is described
Amplified production produces the first cohesive end through USER cleavages.Due to SEQ ID NO:3、SEQ ID NO:Contain CGGU in 4
Fragment, after obtaining the second amplified production by PCR amplification, the di-phosphate ester between USER cleavages the second amplified production G and U
Key, forms the first cohesive end that phosphate group is contained in 5 ' ends, which is conducive to during subsequent reactions
Jointing.
Preferably, the connector contains the second cohesive end, the second viscosity of second cohesive end and amplified production
End complete complementary pairing.Connector is connected by the first cohesive end complementary pairing of the second cohesive end and amplified production, is carried
High joint efficiency.
Preferably, biotin labeling is contained on the connector end opposite with the second cohesive end, and is fixed in advance
On the magnetic bead containing Streptavidin or Avidin.Using the biotin labeling, can very easily will after step B
The library molecule of site containing rs5186 and connector is separated.
It should be noted that when the rs5186 sites to multiple and different samples expand, amplification step is in difference
Separately carried out in reaction system, but the library molecule in the site containing rs5186 of multiple and different samples may be mixed together together
Shi Jinhang is sequenced.When gene tester using the present invention is in same system while to the rs5186 sites of different samples
When being sequenced, since the sequencing primer used in sequencing procedure is identical, it is therefore desirable to use and contain different sequence labels
Connector connect different samples respectively, different sample is distinguished by the sequencing to sequence label, it is more without being carried out to sample
Secondary sequencing can effectively distinguish the sequencing result of different samples.
Contain sequence label on the connector, when multiple tagmeme points to be measured are sequenced in same system, use
Connector containing different sequence labels is connected from the amplified production containing different tagmeme points to be measured, is marked in sequencing procedure by detecting
Sequence is signed, sample can be distinguished in sequencing procedure.
Preferably, the connector includes at least one of connector A, connector B, connector C, connector D；The connector A is by SEQ
ID NO:9 and SEQ ID NO:10 compositions；The connector B is by SEQ ID NO:11 and SEQ ID NO:12 compositions；The connector C
By SEQ ID NO:13 and SEQ ID NO:14 compositions；The connector D is by SEQ ID NO:15 and SEQ ID NO:16 compositions.Its
In, sequence label AATT is contained on the connector A；Contain sequence label TTAA on the connector B；Contain mark on the connector C
Sign sequence C CGG；Contain sequence label GGCC on the connector D.When multiple and different samples are sequenced in same system
When, connected from the amplified production of different samples using the connector containing different sequence labels, marked in sequencing procedure by detecting
Sequence is signed, sample can be distinguished in sequencing procedure；The joint sequence of this programme design is since 5 ' ends are free of phosphate group, no
It can occur from connecting, so as to improve the joint efficiency between connector and amplified production；And the four kinds of labels combined in joint sequence
Sequence base identification during subsequent detection is simple, easily distinguishable；Sequence label length is 4bp, reduces design and detection
Difficulty.
Preferably, the second generation high throughput gene sequencing technology includes but are not limited to connection PCR sequencing PCR or synthesis order-checking
Method.
Preferably, the sequencing primer library molecule in the site containing rs5186 being sequenced in step C is SEQ ID NO:
17.The sequencing primer of this programme design, is connected to rs5186 location proximates region so that in sequencing procedure in sequencing procedure
In, it is only necessary to the sequencing of fewer number can determine that the genotype in rs5186 sites.
Preferably, the phosphorylated modification in 5 ' ends of the sequencing primer.The sequencing primer of this programme is particularly suitable for adopting
With connection PCR sequencing PCR, in sequencing procedure, sequencing probe is connected to the end of sequencing primer 5 '.
The present invention provides 3rd embodiment, a kind of AGTR1 gene testers, with one He of human whole blood genome sample
Human whole blood genome sample two is template, establishes reaction system according to the following steps respectively：
A1, the reaction system for above-mentioned two sample, respectively one amplification AGTR1 genetic fragment of preparation,；Prepare according to the following ratio
Each reaction system：The 1.0 μ L of human whole blood DNA molecular of 50ng/ μ L；2×long Taq Mix（Shenzhen Hua Yinkang Gene sciences have
Limit company produces）10μL；10 μM of 0.4 μ L of the first sense primer；10 μM of 0.4 μ L of the first anti-sense primer；8.2 μ L deionizations
Water；Mix and centrifuge.Then centrifuge tube is placed in PCR instrument, response procedures is set：Continue 5 minutes under the conditions of 94 DEG C；94 DEG C of bars
It is for 20 seconds under part, it is for 20 seconds under the conditions of 57 DEG C, continue 25 seconds under the conditions of 72 DEG C, altogether 30 circulations；Held under the conditions of 72 DEG C
It is 5 minutes continuous；After the completion of PCR reactions, the first amplified production is obtained.Wherein, the first sense primer group is SEQ ID NO:1, first
Anti-sense primer is SEQ ID NO:5；
A2, using the first amplified production of above-mentioned two sample as template, it is each to prepare one and expand the nucleic acid for including rs5186 sites
The reaction system of fragment；Each reaction system is prepared according to the following ratio：The 1.0 μ L of the first amplified production of 100 times of dilution；2×long
Taq Mix 10μL；10 μM of 0.4 μ L of the second sense primer；10 μM of 0.4 μ L of the second anti-sense primer；8.2 μ L deionized waters；It is mixed
It is even and centrifuge.Centrifuge tube is placed in PCR instrument, response procedures are set：Continue 5 minutes under the conditions of 94 DEG C；Continue under the conditions of 94 DEG C
It is for 20 seconds under the conditions of 20 seconds, 57 DEG C, continue 25 seconds under the conditions of 72 DEG C, altogether 35 circulations；Continue 5 minutes under the conditions of 72 DEG C；
After the completion of PCR reactions, the second amplified production is obtained.Wherein, the second sense primer is SEQ ID NO:3, the second anti-sense primer is SEQ
ID NO:7。
B, for the second amplified production of two different samples, it is as follows that cutting-coupled reaction system is prepared respectively：Second expands
Increase production 1 μ L, USER enzyme of thing 20 μ L, T4 ligase buffer solution, 8 μ L, T4 DNA ligase, 2 μ L, 0.2 μm of the advance of ol/mL is fixed
1 μ L of F connectors on magnetic bead.Above-mentioned reaction system is reacted 10 minutes under the conditions of 25 DEG C, and after the completion of reaction, centrifuge tube is placed in
On magnetic frame, separate except supernatant, the TE buffer solutions for adding 50 μ L clean 2 times repeatedly, obtain adsorbing on magnetic bead containing connecing
The library molecule of the nucleic acid fragment of head and rs5186.Wherein, it is connected in the present embodiment with the second amplified production of sample one
Connector is SEQ ID NO:9 and SEQ ID NO:The connector A of 10 compositions, the connector being connected with the second amplified production of sample two are
By SEQ ID NO:11 and SEQ ID NO:The connector B of 12 compositions.
As shown in Figure 1, the library molecule that the library molecule and sample two that are obtained to sample one obtain carries out PAGE glue electrophoresis
Figure detects, and swimming lane 0 is molecular size label in figure, and swimming lane 1 is the library molecule that sample one obtains, and swimming lane 2 obtains for sample two
The library molecule arrived.Occur target stripe near 43bp, be consistent completely with theory expectation library molecule size, illustrate this hair
Bright amplimer group can be very good the nucleic acid fragment in amplification site containing rs5186.
C, the library molecule of the obtained samples one of step B and the library molecule of sample two, point sample to isothiocyano are modified
Load sample on piece, in 37 DEG C of fixed 1h.Using the high throughput gene sequencer Pstar of Shenzhen HYK Gene Technology Co., Ltd.
II A, is sequenced with connecting PCR sequencing PCR, and the rs5186 sites for determining sample one are A, is wild type；The rs5186 positions of sample two
Point is C, is saltant type.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Kang Xin Rui Jiyin health Science and Technology Ltd.
<120>AGTR1 gene detecting kits and detection method
<130>
<160> 17
<170> PatentIn version 3.3
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cctccctgca gtctctatgg gcaattctgc tagtcgctga agtagtcggt 50
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ctacttcagc gactagcaga attgcccata gagactgcag gg 42
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Claims (10)
1. a kind of AGTR1 gene detecting kits, including for carrying out specific amplification to the nucleic acid fragment in the site containing rs5186
Primer sets, it is characterised in that the primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3
Or SEQ ID NO:4, and anti-sense primer SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8.
2. gene detecting kit according to claim 1, it is characterised in that the gene detecting kit, which further includes, to be connect
Head, the connector include at least one of connector A, connector B, connector C, connector D；The connector A is by SEQ ID NO:9 Hes
SEQ ID NO:10 compositions；The connector B is by SEQ ID NO:11 and SEQ ID NO:12 compositions；The connector C is by SEQ ID
NO:13 and SEQ ID NO:14 compositions；The connector D is by SEQ ID NO:15 and SEQ ID NO:16 compositions.
3. gene detecting kit according to claim 1, it is characterised in that the gene detecting kit further includes survey
Sequence primer SEQ ID NO:17, the sequencing primer is used to the library molecule in the site containing rs5186 be sequenced.
4. gene detecting kit according to claim 3, it is characterised in that phosphorus is passed through in 5 ' ends of the sequencing primer
Acidifying modification.
5. a kind of gene tester, it is characterised in that comprise the following steps：
A, using amplimer group, the sample in the site containing rs5186 is expanded, obtains amplified production；It is it is characterized in that, described
Primer sets include sense primer SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:4, and downstream draws
Thing SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7 or SEQ ID NO:8；
B, the jointing on the amplified production, obtains library molecule；
C, the library molecule is sequenced using second generation high throughput gene sequencing technology, determines the gene in rs5186 sites
Type.
6. gene tester according to claim 5, it is characterised in that the step A comprises the following steps：
A1, using the first primer sets expand AGTR1 genetic fragments, obtain the first amplified production；First primer sets include first
Sense primer SEQ ID NO:1 or SEQ ID NO:2, and the first anti-sense primer SEQ ID NO:5 or SEQ ID NO:6；；
A2, using the first amplified production as template, using the second primer sets amplification the site containing rs5186 nucleic acid fragment, obtain the second expansion
Increase production thing；Second primer sets include the second sense primer SEQ ID NO:3 or SEQ ID NO:4, and the second downstream draws
Thing SEQ ID NO:7 or SEQ ID NO:8.
7. gene tester according to claim 6, it is characterised in that further included in the step B to the second amplification
The step of USER enzymes are cut is added in product, second amplified production forms the first cohesive end through USER cleavages.
8. gene tester according to claim 5, it is characterised in that the connector includes connector A, connector B, connector
C, at least one of connector D；The connector A is by SEQ ID NO:9 and SEQ ID NO:10 compositions；The connector B is by SEQ
ID NO:11 and SEQ ID NO:12 compositions；The connector C is by SEQ ID NO:13 and SEQ ID NO:14 compositions；The connector
D is by SEQ ID NO:15 and SEQ ID NO:16 compositions.
9. gene tester according to claim 5, it is characterised in that be used in step C to the site containing rs5186
The sequencing primer that library molecule is sequenced is SEQ ID NO:17.
10. gene tester according to claim 9, it is characterised in that phosphorus is passed through in 5 ' ends of the sequencing primer
Acidifying modification.
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