CN106086190A - Detect reagent set and method that whether pulmonary hypertension related gene undergos mutation - Google Patents
Detect reagent set and method that whether pulmonary hypertension related gene undergos mutation Download PDFInfo
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Abstract
The invention discloses reagent set and method that whether detection pulmonary hypertension related gene undergos mutation.Reagent set disclosed by the invention, being made up of these nine primer sets of BMPR2 PG, ACVRL1 PG, SMAD4 PG, SMAD9 PG, KCNK3 PG, NOTCH3 PG, CAV1 PG, ENG PG and VIP PG, the sequence of each single stranded DNA of these nine primer sets is respectively as shown in sequence 1 330 in sequence table.Experiment proves, the testing result detecting the reagent set whether pulmonary hypertension related gene undergos mutation utilizing the present invention is consistent with the result utilizing high-flux sequence method to carry out detecting, and shows whether the reagent set detection pulmonary hypertension related gene that can utilize the present invention undergos mutation.
Description
Technical field
The present invention relates to biological technical field detects the reagent set whether pulmonary hypertension related gene undergos mutation
And method.
Background technology
Pulmonary hypertension (Pulmonary Arterial Hypertension, PAH) is the disease that a kind of extreme is serious.
Although the application of the new therapy such as some new medicines and gene therapy, live body lung transplantation, interatrial septum fistulation, makes patient 5 in recent years
Year or 10 annual survival rates improve, but the many simply relief of symptoms of its therapeutic scheme, and the process of disease can not be changed with clinical
Consequence.One of them major reason is that the pathogenic mutation of PAH is not also fully apparent from, and the relation of its pathogenesis and clinical phenotypes is also
The clearest and the most definite.Bone morphogenetic protein2 receptor, serotonin, Serotonin Transporter, prostacyclin receptor, prostacyclin synthase, electricity
The potassium channel of pressure gate control, nitric oxide, endothelin-1, changing of the gene coding region such as ETA, B receptor and active oxygen
Becoming all may be relevant with the formation of PAH.Therefore, the understanding above-mentioned protein gene regulated and controled, it will help the most deep recognizes
Know the cause of disease of PAH, prognosis and treatment etc..
Identify that the Disease-causing gene of PAH and the method for sudden change are candidate gene approach the earliest, and the target area of up-to-date rise is (
By early-stage Study identification) high-flux sequence method, but, these methods are time-consuming and spend higher, and application clinically is limited
System.
Summary of the invention
The technical problem to be solved is how to detect pulmonary hypertension related gene (BMPR2 gene, ACVRL1
Gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and VIP gene) whether
Undergo mutation.
For solving above-mentioned technical problem, present invention firstly provides whether detection pulmonary hypertension related gene undergos mutation
Reagent set.
The reagent set whether detection pulmonary hypertension related gene provided by the present invention undergos mutation, by BMPR2-
PG, ACVRL1-PG, SMAD4-PG, SMAD9-PG, KCNK3-PG, NOTCH3-PG, CAV1-PG, ENG-PG and/or VIP-PG group
Become；Described pulmonary hypertension related gene is BMPR2 gene, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 base
Cause, NOTCH3 gene, CAV1 gene, ENG gene and/or VIP gene；
Described BMPR2-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 1-30 in sequence table, wherein, sequence
2a-1 and the single stranded DNA shown in sequence 2a are paired primer, and a is any one natural number in 1 to 15；
Described ACVRL1-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 31-46 in sequence table, wherein, sequence
Row 2b-1 is paired primer with the single stranded DNA shown in sequence 2b, and b is any one natural number in 16 to 23；
Described SMAD4-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 47-64 in sequence table, wherein, sequence
Row 2c-1 is paired primer with the single stranded DNA shown in sequence 2c, and c is any one natural number in 24 to 32；
Described SMAD9-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 65-76 in sequence table, wherein, sequence
Row 2d-1 is paired primer with the single stranded DNA shown in sequence 2d, and d is any one natural number in 33 to 38；
Described KCNK3-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 77-84 in sequence table, wherein, sequence
Row 2e-1 is paired primer with the single stranded DNA shown in sequence 2e, and e is any one natural number in 39 to 42；
Described NOTCH3-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 85-136 in sequence table, wherein,
Sequence 2f-1 and the single stranded DNA shown in sequence 2f are paired primer, and f is any one natural number in 43 to 68；
Described CAV1-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 137-142 in sequence table, wherein, sequence
Row 2g-1 is paired primer with the single stranded DNA shown in sequence 2g, and g is any one natural number in 69 to 71；
Described ENG-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 143-164 in sequence table, wherein, sequence
Row 2h-1 is paired primer with the single stranded DNA shown in sequence 2h, and h is any one natural number in 72 to 82；
Described VIP-PG can be for the primer sets being made up of the single stranded DNA shown in sequence 165-172 in sequence table, wherein, sequence
Row 2i-1 is paired primer with the single stranded DNA shown in sequence 2i, and i is any one natural number in 83 to 86.
In above-mentioned reagent set, described BMPR2-PG is by drawing that the single stranded DNA being combined with BMPR2 gene specific forms
Thing group；
Described ACVRL1-PG is the primer sets being made up of the single stranded DNA being combined with ACVRL1 gene specific；
Described SMAD4-PG is the primer sets being made up of the single stranded DNA being combined with SMAD4 gene specific；
Described SMAD9-PG is the primer sets being made up of the single stranded DNA being combined with SMAD9 gene specific；
Described KCNK3-PG is the primer sets being made up of the single stranded DNA being combined with KCNK3 gene specific；
Described NOTCH3-PG is the primer sets being made up of the single stranded DNA being combined with NOTCH3 gene specific；
Described CAV1-PG is the primer sets being made up of the single stranded DNA being combined with CAV1 gene specific；
Described ENG-PG is the primer sets being made up of the single stranded DNA being combined with ENG gene specific；
Described VIP-PG is the primer sets being made up of the single stranded DNA being combined with VIP gene specific.
In above-mentioned reagent set, described BMPR2-PG can meet following condition: by utilizing described BMPR2-PG to carry out PCR
Expand and the pcr amplification product obtained is carried out order-checking and obtain the full length sequence of BMPR2 gene, the full length sequence of BMPR2 gene
Can obtain by the sequence of the pcr amplification product of described BMPR2-PG is carried out splicing；
Described ACVRL1-PG can meet following condition: by utilizing described ACVRL1-PG to carry out PCR amplification and to obtaining
Pcr amplification product carry out order-checking and obtain the full length sequence of ACVRL1 gene, the full length sequence of ACVRL1 gene can be by by institute
The sequence of the pcr amplification product stating ACVRL1-PG carries out splicing and obtains；
Described SMAD4-PG can meet following condition: by utilizing described SMAD4-PG to carry out PCR amplification and to obtaining
Pcr amplification product carries out order-checking and obtains the full length sequence of SMAD4 gene, and the full length sequence of SMAD4 gene can be by by described
The sequence of the pcr amplification product of SMAD4-PG carries out splicing and obtains；
Described SMAD9-PG can meet following condition: by utilizing described SMAD9-PG to carry out PCR amplification and to obtaining
Pcr amplification product carries out order-checking and obtains the full length sequence of SMAD9 gene, and the full length sequence of SMAD9 gene can be by by described
The sequence of the pcr amplification product of SMAD9-PG carries out splicing and obtains；
Described KCNK3-PG can meet following condition: by utilizing described KCNK3-PG to carry out PCR amplification and to obtaining
Pcr amplification product carries out order-checking and obtains the full length sequence of KCNK3 gene, and the full length sequence of KCNK3 gene can be by by described
The sequence of the pcr amplification product of KCNK3-PG carries out splicing and obtains；
Described NOTCH3-PG can meet following condition: by utilizing described NOTCH3-PG to carry out PCR amplification and to obtaining
Pcr amplification product carry out order-checking and obtain the full length sequence of NOTCH3 gene, the full length sequence of NOTCH3 gene can be by by institute
The sequence of the pcr amplification product stating NOTCH3-PG carries out splicing and obtains；
Described CAV1-PG can meet following condition: by utilizing described CAV1-PG to carry out PCR amplification and to the PCR obtained
Amplified production carries out order-checking and obtains the full length sequence of CAV1 gene, and the full length sequence of CAV1 gene can be by by described CAV1-PG
The sequence of pcr amplification product carry out splicing and obtain；
Described ENG-PG can meet following condition: by utilizing described ENG-PG carry out PCR amplification and expand the PCR obtained
Volume increase thing carries out order-checking and obtains the full length sequence of ENG gene, and the full length sequence of ENG gene can be by the PCR by described ENG-PG
The sequence of amplified production carries out splicing and obtains；
Described VIP-PG can meet following condition: by utilizing described VIP-PG carry out PCR amplification and expand the PCR obtained
Volume increase thing carries out order-checking and obtains the full length sequence of VIP gene, and the full length sequence of VIP gene can be by the PCR by described VIP-PG
The sequence of amplified production carries out splicing and obtains.
Described PCR amplification can be all the PCR amplification carried out for template with corresponding full-length gene.
In above-mentioned reagent set, in described reagent set, the molal quantity ratio of each single stranded DNA can be according to actually detected
Sample is adjusted, and in described reagent set, the molal quantity liquid of each single stranded DNA all can be identical.
Each single stranded DNA in described reagent set all can independent packaging, it is possible to be packaged together；Also can by therein in pairs
Primer is individually packed, it is also possible to be packaged together by paired at least two therein primer；Genes of interest also dependent on detection
Difference the primer sets for same gene is the most individually packed or one will be packaged in for the primer sets of several genes
Rising (will described BMPR2-PG, described ACVRL1-PG, described SMAD4-PG, described SMAD9-PG, described KCNK3-PG, described
NOTCH3-PG, described CAV1-PG, described ENG-PG and described VIP-PG pack respectively or at least two therein are packed
Together).
For solving above-mentioned technical problem, present invention also offers whether the described pulmonary hypertension related gene of detection dashes forward
The system become.
The system whether detection pulmonary hypertension related gene provided by the present invention undergos mutation, including described complete examination
Agent and Y1；Described Y1 is to carry out the reagent required for PCR amplification and/or instrument.
In said system, described system can be to include described reagent set and the described reagent carried out required for PCR amplification
Reagent or test kit.Reagent required for the described PCR of carrying out amplification can be 2 × KAPA HiFi HotStart ReadyMix.
2 × KAPA HiFi HotStart ReadyMix is KAPA Biosystems product.
For solving above-mentioned technical problem, present invention also offers whether the described pulmonary hypertension related gene of detection dashes forward
The method become.
Detection provided by the present invention or auxiliary detect the method whether described pulmonary hypertension related gene undergos mutation,
Including: with the genomic DNA of people's Isolated-lung tissue to be measured as template, utilize described reagent set to carry out multiplexed PCR amplification, obtain
Pcr amplification product；BMPR2 gene (Serial No. NM_ in NCBI by the sequence of described pcr amplification product Yu wild type
001204), ACVRL1 gene (Serial No. NM_000020 in NCBI), the SMAD4 gene (Serial No. in NCBI
NM_005359), SMAD9 gene (Serial No. NM_001127217 in NCBI), the KCNK3 gene (sequence in NCBI
Number be NM_002246), NOTCH3 gene (Serial No. NM_000435 in NCBI), the CAV1 gene (sequence in NCBI
Row number are NM_001172895), ENG gene (Serial No. NM_000118 in NCBI) and VIP gene be (in NCBI
Serial No. NM_003381) in the sequence of corresponding gene compare, determine whether described pulmonary hypertension related gene occurs
Sudden change.
In said method, described multiplexed PCR amplification can be carried out under the annealing temperature of 55 DEG C.Described multiplexed PCR amplification can
Anneal 30 seconds under the annealing temperature of 55 DEG C.The reaction condition of described multiplexed PCR amplification is concretely: 98 DEG C of 5min；98℃
30sec, 55 DEG C of 30sec, 72 DEG C of 45sec, circulate 20 times；72℃5min.
In said method, described multiplexed PCR amplification can be carried out in reaction system 1；Described reaction system 1 can be treated by described
Survey the genomic DNA of human blood, described reagent set, 2 × KAPA HiFi HotStart ReadyMix and water composition.Described
Described in reaction system 1, the concentration of the genomic DNA of people's Isolated-lung tissue to be measured can be 50ng/20 μ l, described reagent set
Concentration can be 12.5mM.
Said method may also include that the product to described multiplex PCR carries out A-tailing, jointing and amplification successively.
For solving above-mentioned technical problem, present invention also offers following arbitrary application:
The application in preparation prediction or auxiliary prediction pulmonary hypertension product of X1, described reagent set；
The application in prediction or auxiliary prediction pulmonary hypertension of X2, described reagent set；
In preparation detection or auxiliary, X3, described reagent set detect whether described pulmonary hypertension related gene undergos mutation
Application in product；
X4, described reagent set are in whether detection or the auxiliary described pulmonary hypertension related gene of detection undergo mutation
Application；
The application in preparation prediction or auxiliary prediction pulmonary hypertension product of X5, described system；
The application in prediction or auxiliary prediction pulmonary hypertension of X6, described system；
In preparation detection or auxiliary, X7, described system detect whether described pulmonary hypertension related gene undergos mutation product
In application；
X8, described system detect answering during whether described pulmonary hypertension related gene undergos mutation in detection or auxiliary
With；
The application in prediction or auxiliary prediction pulmonary hypertension of X9, described method.
For solving above-mentioned technical problem, present invention also offers prediction or whether auxiliary prediction people suffers from pulmonary hypertension
Method.
Prediction provided by the present invention or auxiliary predict the method whether object to be measured suffers from pulmonary hypertension, including: profit
The method whether undergone mutation with described detection described pulmonary hypertension related gene determines described pulmonary hypertension related gene
Whether undergo mutation, the object to be measured that described pulmonary hypertension related gene is undergone mutation suffers from the risk of pulmonary hypertension and is higher than
Or the object to be measured that candidate does not undergos mutation higher than described pulmonary hypertension related gene.
It is demonstrated experimentally that utilize the reagent set whether the detection pulmonary hypertension related gene of the present invention undergos mutation to three
BMPR2, ACVRL1, SMAD4, SMAD9, KCNK3, NOTCH3, CAV1, ENG and VIP base in individual patients with pulmonary hypertension family
The sequence of cause detects, and the 1038th of the BMPR2 gene of the patients with pulmonary hypertension in discovery family 1 by thymus pyrimidine
(T) sudden change is for cytosine (C), and other genes are not all undergone mutation, rather than these nine genes of patients with pulmonary hypertension are the most not
Undergo mutation；The 1042nd of BMPR2 gene in family 2 is suddenlyd change for adenine (A) by guanine (G), and other genes are equal
Do not undergo mutation, rather than these nine genes of patients with pulmonary hypertension are not all undergone mutation；Pulmonary hypertension in non-family 3 is suffered from
The 1450th of the ACVRL1 gene of person is suddenlyd change for thymus pyrimidine (T) by cytosine (C), and other genes are not all undergone mutation,
Rather than these nine genes of patients with pulmonary hypertension are not all undergone mutation.These results are examined with utilizing high-flux sequence method
The result surveyed is consistent.Showing, the reagent set whether the detection pulmonary hypertension related gene of the present invention undergos mutation can be used
Detect whether pulmonary hypertension related gene undergos mutation.
Therefore, applicant, for pulmonary hypertension related gene, establishes detection pulmonary hypertension phase based on multiplex PCR
The method whether correlation gene undergos mutation.Whether target gene undergos mutation and then can examination PAH to utilize the method can determine that
Known pathogenic mutation, quickly makes gene diagnosis for patient.Can also collect for by the known pathogenic mutation of investigation
Know the PAH family that sudden change is negative, set up queue, carry out the gene sequencing of full exon, search with disease be divided into from cause a disease new
Sudden change.
In view of merging BMPR2, ACVRL1, SMAD4, SMAD9, KCNK3, NOTCH3, CAV1, ENG, VIP gene mutation
The poor prognosis that PAH is possible, tackles PAH patient, especially heritability PAH, idiopathic PAH and/or the patient of merging HHT and parent
Belong to row detection in Gene Mutation, for making a definite diagnosis pathological changes ahead of time, provide the prediction of initiation potential degree significant for asymptomatic relatives, involutory
And potential the patient suddenlyd change, it is proposed that every 3 years check ultrasoundcardiograms, in conjunction with the close observation of clinical symptoms, the early stage to PAH
Diagnosis and intervening is of great importance improving clinical prognosis.To child PAH patient, also should actively row gene test with as early as possible for sick
The clearly offer foundation of cause, detection parents, to find to carry the high risk person of Disease-causing gene sudden change, measure father and mother more simultaneously
The risk that row fertility offspring is ill.
The experiment proves that, present invention discover that a kind of new pulmonary hypertension Disease-causing gene mutational site, itself and lung
Arterial hypertension is correlated with, by detecting whether as this mutational site, it was predicted that whether sample to be tested suffers from pulmonary hypertension, and it can be made
For treating the target spot of pulmonary hypertension, provide new therapy approach for study of lung arterial hypertension medicine in the future.
Accompanying drawing explanation
Fig. 1 is three patients with pulmonary hypertension sample pedigree charts.Wherein, figure A, figure B and figure C are respectively pulmonary hypertension trouble
Person's sample family 1, family 2 and family 3, patient shown in arrow is pulmonary hypertension proband in this family.
Fig. 2 is the sequence verification result of patients with pulmonary hypertension DNA in three patients with pulmonary hypertension sample familys.Its
In, A is the 1038th neighbouring DNA sequencing result of patients with pulmonary hypertension BMPR2 gene in family 1, and B is lung in family 2
1042nd neighbouring DNA sequencing result of arterial hypertension patient's BMPR2 gene, C is patients with pulmonary hypertension in family 3
1450th neighbouring DNA sequencing result of ACVRL1 gene.
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for explaining
The bright present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
2 × KAPA HiFi HotStart ReadyMix in following embodiment is KAPA Biosystems product, produces
Product catalog number (Cat.No.) is KK2601.
Embodiment 1, the reagent set detection pulmonary artery utilizing detection pulmonary hypertension related gene whether to undergo mutation are high
Whether pressure related gene undergos mutation
One, the preparation of the reagent set whether detection pulmonary hypertension related gene undergos mutation
The detection reagent set whether undergone mutation of pulmonary hypertension related gene, by BMPR2-PG, ACVRL1-PG,
SMAD4-PG, SMAD9-PG, KCNK3-PG, NOTCH3-PG, CAV1-PG, ENG-PG and VIP-PG form；
BMPR2-PG is the primer sets being made up of the single stranded DNA shown in sequence 1-30 in sequence table, wherein, sequence 2a-1 with
Single stranded DNA shown in sequence 2a is paired primer, and a is the natural number in 1-15；BMPR2-PG can meet following condition: by profit
Carry out PCR amplification with BMPR2-PG with BMPR2 gene for template and the pcr amplification product obtained is carried out order-checking can obtain
The full length sequence of BMPR2 gene, the full length sequence of BMPR2 gene can be by entering the sequence of the pcr amplification product of BMPR2-PG
Row splicing obtains；
ACVRL1-PG is the primer sets being made up of the single stranded DNA shown in sequence 31-46 in sequence table, wherein, sequence 2b-1
Being paired primer with the single stranded DNA shown in sequence 2b, b is the natural number in 16-23；ACVRL1-PG can meet following condition: logical
Cross and utilize ACVRL1-PG carry out PCR amplification with ACVRL1 gene for template and check order permissible to the pcr amplification product obtained
Obtaining the full length sequence of ACVRL1 gene, the full length sequence of ACVRL1 gene can be by by the pcr amplification product of ACVRL1-PG
Sequence carries out splicing and obtains；
SMAD4-PG is the primer sets being made up of the single stranded DNA shown in sequence 47-64 in sequence table, wherein, sequence 2c-1
Being paired primer with the single stranded DNA shown in sequence 2c, c is the natural number in 24-32；SMAD4-PG can meet following condition: logical
Cross and utilize SMAD4-PGG carry out PCR amplification with SMAD4 gene for template and check order permissible to the pcr amplification product obtained
Obtaining the full length sequence of SMAD4 gene, the full length sequence of SMAD4 gene can be by the sequence by the pcr amplification product of SMAD4-PG
Row carry out splicing and obtain；
SMAD9-PG is the primer sets being made up of the single stranded DNA shown in sequence 65-76 in sequence table, wherein, sequence 2d-1
Being paired primer with the single stranded DNA shown in sequence 2d, d is the natural number in 33-38；SMAD9-PG can meet following condition: logical
Cross and utilize SMAD9-PG carry out PCR amplification with SMAD9 gene for template and the pcr amplification product obtained is carried out order-checking can obtain
Obtaining the full length sequence of SMAD9 gene, the full length sequence of SMAD9 gene can be by the sequence by the pcr amplification product of SMAD9-PG
Carry out splicing to obtain；
KCNK3-PG is the primer sets being made up of the single stranded DNA shown in sequence 77-84 in sequence table, wherein, sequence 2e-1
Being paired primer with the single stranded DNA shown in sequence 2e, e is the natural number in 39-42；KCNK3-PG can meet following condition: logical
Cross and utilize KCNK3-PG carry out PCR amplification with KCNK3 gene for template and the pcr amplification product obtained is carried out order-checking can obtain
Obtaining the full length sequence of KCNK3 gene, the full length sequence of KCNK3 gene can be by the sequence by the pcr amplification product of KCNK3-PG
Carry out splicing to obtain；
NOTCH3-PG is the primer sets being made up of the single stranded DNA shown in sequence 85-136 in sequence table, wherein, sequence 2f-
1 with the single stranded DNA shown in sequence 2f be paired primer, f is the natural number in 43-68；NOTCH3-PG can meet following condition:
By utilize NOTCH3-PG with NOTCH3 gene for template carry out PCR amplification and the pcr amplification product obtained is carried out order-checking can
To obtain the full length sequence of NOTCH3 gene, the full length sequence of NOTCH3 gene can be by by the pcr amplification product of NOTCH3-PG
Sequence carry out splicing and obtain；
CAV1-PG is the primer sets being made up of the single stranded DNA shown in sequence 137-142 in sequence table, wherein, sequence 2g-1
Being paired primer with the single stranded DNA shown in sequence 2g, g is the natural number in 69-71；CAV1-PG can meet following condition: passes through
CAV1-PG is utilized to carry out PCR amplification with CAV1 gene for template and the pcr amplification product obtained is carried out order-checking can obtain
The full length sequence of CAV1 gene, the full length sequence of CAV1 gene can be by spelling the sequence of the pcr amplification product of CAV1-PG
Connect and obtain；
ENG-PG is the primer sets being made up of the single stranded DNA shown in sequence 143-164 in sequence table, wherein, sequence 2h-1
Being paired primer with the single stranded DNA shown in sequence 2h, h is the natural number in 72-82；ENG-PG can meet following condition: passes through
ENG-PG is utilized to carry out PCR amplification with ENG gene for template and the pcr amplification product obtained is carried out order-checking can obtain ENG
The full length sequence of gene, the full length sequence of ENG gene can be by carrying out splicing by the sequence of the pcr amplification product of ENG-PG
Arrive；
VIP-PG is the primer sets being made up of the single stranded DNA shown in sequence 165-172 in sequence table, wherein, sequence 2i-1
Being paired primer with the single stranded DNA shown in sequence 2i, i is the natural number in 83-86；VIP-PG can meet following condition: passes through
VIP-PG is utilized to carry out PCR amplification with VIP gene for template and the pcr amplification product obtained is carried out order-checking can obtain VIP
The full length sequence of gene, the full length sequence of VIP gene can be by carrying out splicing by the sequence of the pcr amplification product of VIP-PG
Arrive.
Each single stranded DNA independent packaging in the reagent set whether detection pulmonary hypertension related gene undergos mutation, each list
The molal quantity of chain DNA is the most identical.
Two, the detection whether pulmonary hypertension related gene undergos mutation
Three patients with pulmonary hypertension sample pedigree charts as it is shown in figure 1, in Fig. 1 A be the pedigree chart of family 1, B is family 2
Pedigree chart, C is the pedigree chart of family 3.Wherein family 1 has three patients, remaining the most non-patients with pulmonary hypertension, other two
Distributing patients with pulmonary hypertension (i.e. in family 2 in patient and family 3 patient), its father and mother are the most non-patients with pulmonary hypertension.
Utilize the reagent set whether the detection pulmonary hypertension related gene of step one undergos mutation by the following method
Respectively whether everyone pulmonary hypertension related gene of this family is undergone mutation and detects:
1, the extraction of genomic DNA:
Take sample anticoagulated whole blood to be detected, utilize QIAGEN company DNA extraction kit to extract and obtain sample base to be detected
Because of group DNA；
2, multiplexed PCR amplification
Multiplexed PCR amplification reaction system is as follows: sample genomic dna 50ng to be detected；The detection pulmonary artery of step one is high
Reagent set that whether pressure related gene undergos mutation (the denseest in this reaction system of all single stranded DNAs in this reagent set
Degree is 12.5mM)；2×KAPA HiFi HotStart ReadyMix 10μl；ddH2O mends to 20 μ l.
The condition of multiplexed PCR amplification reaction is: 98 DEG C of denaturations 5min；98 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72
DEG C extend 45sec, circulate 20 times；72 DEG C of extension 5min the most last.
The PCR primer purification kit that the pcr amplification product obtained utilizes QIAGEN company is purified, and obtains purification
After pcr amplification product.
3, library (for secondary order-checking, this step is that in secondary order-checking, before machine prepared by sample) is set up
3.1A-tailing
Reaction system is: the pcr amplification product 8 μ l, 10 × blue buffer after purification of step 2 (Enzymatics,
B011) 1.25 μ l, dATP (1mM) 2.5 μ l (Enzymatics company), Klenow (3 '-5 ' exo-) (Enzymatics company,
P7010-HC-L) 0.75 μ l, cumulative volume is 12.5 μ l.
Above-mentioned reaction system is hatched 30min under 37 degree, in reaction system, then adds 25 μ l AMPure XP magnetic
(Beckman Coulter, A63880 are a kind of Nucleic acid purification kits being obtained in that high quality DNA product to pearl；After purification
Nucleic acid purity high, salt-free ion residues, it is not necessary to centrifugal or filter) sort, the pcr amplification product of tailing of succeeding,
The pcr amplification product of success tailing is dissolved in 8 μ l and dissolves the pcr amplification product obtaining tailing in buffer.
3.2 joints connect
Reaction system: pcr amplification product 7.5 μ l, the 2 × Rapid ligation buffer of the tailing of step 3.1
(Enzymatics company, B101) 12.5 μ l, Adapters (10 μMs) (Adptor sequence: InPEA1,
GATCGGAAGAGCACACGTCT；InPEA2, ACACTCTTTCCCTACACGACGCTCTTCCGATCT, use after annealing)
2.5 μ l, T4DNA Ligase (Enzymatics company, L603-HC-L) 2.5 μ l, cumulative volume is 25 μ l.
Above-mentioned reaction system is hatched 15min under 20 degree, in reaction system, then adds 25 μ l AMPure XP magnetic
(Beckman Coulter, A63880 are a kind of Nucleic acid purification kits being obtained in that high quality DNA product to pearl；After purification
Nucleic acid purity high, salt-free ion residues, it is not necessary to centrifugal or filter) sort, the PCR amplification obtaining joint successful connection is produced
Thing, is dissolved in the pcr amplification product of joint successful connection 23 μ l and dissolves the pcr amplification product obtaining jointing in buffer.
Expand before 3.3
Reaction system: pcr amplification product 11.5 μ l, PE 1.0 (25 μMs) (PE 1.0 sequence of the jointing of step 3.2
Row: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT) 0.5 μ l, PE 2.0+
Index (25 μMs) 0.5 μ l, 2 × KAPA HiFi HotStart ReadyMix 12.5 μ l, cumulative volume is 25 μ l.Wherein, PE
The sequence of each single stranded DNA in 2.0+index is as follows:
InPE-2.0-1:
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-2:
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-3:
CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-4:
CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-5:
CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-6:
CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-7:
CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-8:
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-9:
CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-10:
CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-11:
CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-12:
CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-13:
CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-14:
CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-15:
CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-16:
CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-17:
CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-18:
CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-19:
CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
InPE-2.0-20:
CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Reaction condition is: 98 DEG C of denaturations 2min；98 DEG C of degeneration 30sec, 65 DEG C of annealing 30sec, 72 DEG C extend 45sec,
Circulate 16 times；72 DEG C of extension 5min the most last.
React and the product obtained carries out after terminating electrophoresis detection and reclaims test kit with the gel of QIAGEN company carrying out
Gel reclaims, by reclaim product carry out quantitatively (concentration of the recovery product DNA that order-checking is required satisfied is 10-20ng/ μ l) laggard
The secondary order-checking of row ILUMINA Hiseq40000 (ILUMINA company secondary order-checking platform).
Sequencing result shows that this recovery product has obtained BMPR2 gene, ACVRL1 gene, SMAD4 base through order-checking splicing
Cause, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and the total length sequence of these 9 genes of VIP gene
Row, by these sequences and the BMPR2 gene (Serial No. NM_001204 in NCBI) of wild type, ACVRL1 gene (
Serial No. NM_000020 in NCBI), SMAD4 gene (Serial No. NM_005359 in NCBI), SMAD9 gene
(Serial No. NM_001127217 in NCBI), KCNK3 gene (Serial No. NM_002246 in NCBI),
NOTCH3 gene (Serial No. NM_000435 in NCBI), CAV1 gene (Serial No. NM_ in NCBI
001172895), ENG gene (Serial No. NM_000118 in NCBI) and the VIP gene (Serial No. in NCBI
NM_003381) in, the sequence of corresponding gene is compared, and determines whether each pulmonary hypertension related gene of sample to be detected is sent out
Raw sudden change.
Result shows, the 1038th of the BMPR2 gene of three patients with pulmonary hypertension in family 1 by thymus pyrimidine
(T) sudden change is for cytosine (C), causes the 347th, BMPR2 protein by cysteine mutation for arginine；These three lung
The ACVRL1 gene of arterial hypertension patient, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene,
The DNA sequence of ENG gene and VIP gene is not all undergone mutation.The BMPR2 base of other non-patients with pulmonary hypertension in family 1
Cause, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and VIP
The DNA sequence of gene is not all undergone mutation.
In family 2, the 1042nd of the BMPR2 gene of patients with pulmonary hypertension is suddenlyd change for adenine by guanine (G)
(A), cause the 348th, BMPR2 protein by valine mutation for isoleucine；The ACVRL1 gene of this patient, SMAD4 base
The DNA sequence of cause, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and VIP gene does not all occur
Sudden change.The BMPR2 gene of the father and mother of this patient, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3
The DNA sequence of gene, CAV1 gene, ENG gene and VIP gene is not all undergone mutation.
In family 3, the 1450th of the ACVRL1 gene of patients with pulmonary hypertension is suddenlyd change in order to thymus is phonetic by cytosine (C)
Pyridine (T), causes the 488th, ACVRL1 protein to be suddenlyd change for tryptophan by arginine；The BMPR2 gene of this patient, SMAD4 base
The DNA sequence of cause, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and VIP gene does not all occur
Sudden change.The BMPR2 gene of the father and mother of this patient, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3
The DNA sequence of gene, CAV1 gene, ENG gene and VIP gene is not all undergone mutation.
It addition, utilize existing high-flux sequence method, (genes of interest capture combines secondary sequence measurement, Beijing step base
Promise Gene science Co., Ltd completes) to the patients with pulmonary hypertension in three patients with pulmonary hypertension sample familys and non-
The BMPR2 gene of patients with pulmonary hypertension, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 base
Cause, CAV1 gene, ENG gene and VIP gene check order, this high-flux sequence result show in these three family every each and every one
The BMPR2 gene of body, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene,
With the utilization utilizing the present invention, the full length sequence of ENG gene and VIP gene detects whether pulmonary hypertension related gene dashes forward
The full length sequence of these 9 genes that the reagent set detection become obtains is identical.
By the 1038th the both sides design primer at BMPR2 gene, utilize this primer respectively to three lungs in family 1
The genomic DNA of arterial hypertension patient carries out PCR amplification, then checks order PCR primer, it was found that three pulmonary artery
The 1038th of the BMPR2 gene of high pressure patient is suddenlyd change for cytosine (C) (Fig. 2) by thymus pyrimidine (T), with the above results one
Cause.
By the 1042nd the both sides design primer at BMPR2 gene, utilize this primer respectively to the pulmonary artery in family 2
The genomic DNA of high pressure patient carries out PCR amplification, then checks order PCR primer, it was found that this pulmonary hypertension is suffered from
The 1042nd of the BMPR2 gene of person is suddenlyd change for adenine (A) (Fig. 2) by guanine (G), consistent with the above results.
By the 1450th the both sides design primer at ACVRL1 gene, this primer is utilized respectively the lung in family 3 to be moved
The genomic DNA of arteries and veins high pressure patient carries out PCR amplification, then checks order PCR primer, it was found that this pulmonary hypertension
The 1450th of the ACVRL1 gene of patient is suddenlyd change for thymus pyrimidine (T) (Fig. 2) by cytosine (C), consistent with the above results.
Claims (10)
1. the detection reagent set whether undergone mutation of pulmonary hypertension related gene, by BMPR2-PG, ACVRL1-PG,
SMAD4-PG, SMAD9-PG, KCNK3-PG, NOTCH3-PG, CAV1-PG, ENG-PG and/or VIP-PG form；Described pulmonary artery
High pressure phase correlation gene be BMPR2 gene, ACVRL1 gene, SMAD4 gene, SMAD9 gene, KCNK3 gene, NOTCH3 gene,
CAV1 gene, ENG gene and/or VIP gene；
Described BMPR2-PG is the primer sets being made up of the single stranded DNA shown in sequence 1-30 in sequence table；
Described ACVRL1-PG is the primer sets being made up of the single stranded DNA shown in sequence 31-46；
Described SMAD4-PG is the primer sets being made up of the single stranded DNA shown in sequence 47-64；
Described SMAD9-PG is the primer sets being made up of the single stranded DNA shown in sequence 65-76；
Described KCNK3-PG is the primer sets being made up of the single stranded DNA shown in sequence 77-84；
Described NOTCH3-PG is the primer sets being made up of the single stranded DNA shown in sequence 85-136；
Described CAV1-PG is the primer sets being made up of the single stranded DNA shown in sequence 137-142；
Described ENG-PG is the primer sets being made up of the single stranded DNA shown in sequence 143-164；
Described VIP-PG is the primer sets being made up of the single stranded DNA shown in sequence 165-172.
Reagent set the most according to claim 1, it is characterised in that: the molal quantity of each single stranded DNA in described reagent set
The most identical.
3. the detection system whether undergone mutation of pulmonary hypertension related gene, including reagent set described in claim 1 or 2 and
Y1；Described Y1 is to carry out the reagent required for PCR amplification and/or instrument.
System the most according to claim 3, it is characterised in that carrying out the reagent required for PCR amplification described in: is 2 × KAPA
HiFi HotStart ReadyMix。
5. detection or auxiliary test right require the method that described in 1, whether pulmonary hypertension related gene undergos mutation, including:
With the genomic DNA of people's Isolated-lung tissue to be measured as template, reagent set described in claim 1 or 2 is utilized to carry out multiplex PCR expansion
Increase, obtain pcr amplification product；By the sequence of described pcr amplification product and BMPR2 gene, ACVRL1 gene, SMAD4 gene,
In SMAD9 gene, KCNK3 gene, NOTCH3 gene, CAV1 gene, ENG gene and VIP gene, the sequence of corresponding gene is carried out
Comparison, determines whether described pulmonary hypertension related gene undergos mutation；
Described multiplexed PCR amplification is carried out under the annealing temperature of 55 DEG C.
Method the most according to claim 5, it is characterised in that: described multiplexed PCR amplification moves back under the annealing temperature of 55 DEG C
Fire 30 seconds.
7. according to the method described in claim 5 or 6, it is characterised in that: described multiplexed PCR amplification is carried out in reaction system 1；
Described reaction system 1 by reagent set described in the genomic DNA of described people's Isolated-lung tissue to be measured, claim 1 or 2,2 ×
KAPA HiFi HotStart ReadyMix and water composition.
Method the most according to claim 7, it is characterised in that: described in described reaction system 1, the concentration of reagent set is
12.5mM。
The most following arbitrary application:
The application in preparation prediction or auxiliary prediction pulmonary hypertension product of the reagent set described in X1, claim 1 or 2；
The application in prediction or auxiliary prediction pulmonary hypertension of the reagent set described in X2, claim 1 or 2；
Reagent set described in X3, claim 1 or 2 requires pulmonary hypertension described in 1 in preparation detection or auxiliary test right
Whether related gene undergos mutation the application in product；
In detection or auxiliary test right, reagent set described in X4, claim 1 or 2 requires that described in 1, pulmonary hypertension is correlated with
Gene whether undergo mutation in application；
The application in preparation prediction or auxiliary prediction pulmonary hypertension product of the system described in X5, claim 5；
The application in prediction or auxiliary prediction pulmonary hypertension of the system described in X6, claim 5；
System described in X7, claim 5 requires pulmonary hypertension related gene described in 1 in preparation detection or auxiliary test right
Whether undergo mutation the application in product；
Whether system described in X8, claim 5 requires pulmonary hypertension related gene described in 1 in detection or auxiliary test right
Application in undergoing mutation；
Arbitrary described method application in prediction or auxiliary prediction pulmonary hypertension in X9, claim 5-8.
10. prediction or auxiliary predict the method whether object to be measured suffers from pulmonary hypertension, including: utilize in claim 5-8
Arbitrary described method determines whether pulmonary hypertension related gene described in claim 1 undergos mutation, and described pulmonary artery is high
The pressure object to be measured undergone mutation of related gene suffer from the risk of pulmonary hypertension higher than or candidate higher than described pulmonary hypertension
The object to be measured that related gene is not undergone mutation.
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| CN108753833A (en) * | 2018-05-28 | 2018-11-06 | 上海海洋大学 | The preparation method of zebra fish notch3 gene mutation bodies |
| CN109797212A (en) * | 2019-01-30 | 2019-05-24 | 中国人民解放军总医院 | A method of/the tumor susceptibility gene that causes a disease of detection pulmonary hypertension |
| CN110669837A (en) * | 2019-09-27 | 2020-01-10 | 广州医科大学附属第一医院（广州呼吸中心） | Caveolin-1 gene probe group for detecting pulmonary hypertension and application thereof |
| CN110846397A (en) * | 2019-09-27 | 2020-02-28 | 广州医科大学附属第一医院（广州呼吸中心） | SMAD9 gene probe set for detecting pulmonary hypertension and application thereof |
| CN110846398A (en) * | 2019-09-27 | 2020-02-28 | 广州医科大学附属第一医院（广州呼吸中心） | BMPR2 gene probe set for detecting pulmonary hypertension and application thereof |
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| CN115806998A (en) * | 2022-11-24 | 2023-03-17 | 百世诺（北京）医疗科技有限公司 | Pulmonary hypertension variant gene NOTCH3 and application thereof |
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