CN108342466A - A kind of high-flux sequence method and its application of psychoneural class pharmaceutical relevant gene - Google Patents
A kind of high-flux sequence method and its application of psychoneural class pharmaceutical relevant gene Download PDFInfo
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Abstract
The invention belongs to psychoneural genoid detection fields, and in particular to a kind of high-flux sequence method and its application of psychoneural class pharmaceutical relevant gene.The present invention provides a kind of method and its application of psychoneural class pharmaceutical relevant gene high-flux sequence, this method includes the acquisition of genomic DNA, target gene multiplexed PCR amplification reacts and product purification, joint sequence PCR reactions and product purification, library detection and high-flux sequence and etc., this method can 29 SNP sites of disposable pair and relevant 20 genes of Nervous and mental diseases medication be detected and can cover antipsychotic, depression, antianxiety disease, anti-epileptic, parkinsonism, mostly dynamic obstacle, the one two wires overwhelming majority drug such as pain, to greatest extent guidance is provided for the medication of patient.
Description
Technical field
The invention belongs to psychoneural genoid detection fields, and in particular to a kind of psychoneural class pharmaceutical relevant gene
High-flux sequence method and its application.
Background technology
Currently, great change has occurred in human diseases spectrum, and neuropsychiatric disease is more and more forward in human diseases pedigree,
And the increasingly increased work that is faced with people of its incidence and life stress are also constantly increasing.At present in China's spirit
Disease accounts for about 20.8% that disease is always born, is expected to increase to 25% to the year two thousand twenty.In June, 2009,《Lancet》Magazine is delivered
Epidemiological survey to Chinese four province's phrenoblabias is as a result, investigation is shown, there are about 1.73 hundred million people to suffer from different type for China
Phrenoblabia.And as aging crowd is continuously increased, the nervous system diseases such as apoplexy, Alzheimer disease, Parkinson's disease
Incidence and illness rate also gradually increasing.
On the other hand, although the therapeutic modality of mental disease is also developed it will be recognized that drug therapy is still mesh
The main therapy of preceding overwhelming majority mental disease and means.Mental disease generally requires the continued treatment under drug control,
But there is potential harmful effects for patient's Long-term taking medicine.Patient's treated and saved to antipsychotics in wide clinical application
Meanwhile adverse drug reaction caused by antipsychotics becomes increasingly conspicuous due to largely using in recent years, is caused to patient
Significant impact.
Antipsychotics mechanism of action is it has a blocking effect to some receptors, and the adverse reaction generated also with this
Mechanism of action is related.Such as：1. the blocking to histamine receptor, the mainly blocking to H1 receptors can generate calm and weight
Increased adverse reaction；2. the blocking to adrenergic receptor can generate sedation and orthostatic mainly to 1 receptors of α
The adverse reaction of low blood pressure, tachycardia, hypogona dism；3. to the blocking effect of cholinergic recepter, mainly to M1 receptors
Blocking, the adverse reactions such as dry, constipation, intestinal obstruction, dysuria can be generated；4. the blocking to dopamine receptor can generate
The extrapyramidal symptoms and the horizontal raised adverse reaction of prolactin(PRL and the most important adverse reaction of this class drug.Antipsychotic
Drug-induced adverse reaction is with nervous system adverse reaction rate highest；The adverse reaction of nervous system is to be anti-outside centrum
Answer incidence highest.It is noted that acute dystonia caused by the extrapyramidal symptoms can cause dysphagia, it is easy
Patient is caused to suffocate, or even dead.Research also shows that adverse drug reaction caused by antipsychotics can betide any year
The crowd of age section, but the blood plasma of different age group patient is different from drug binding ability, medicament metabolism ability and drainage rate,
Cause probability, the severity of initiation adverse reaction different.
In terms of drug metabolism, liver is the maximum body of gland of human body, and containing a large amount of active metabolism enzyme, it is not only in food
Metabolic process in play an important role, while be also many chemical substances and drug important metabolic organ.Chemical substance or
After drug is absorbed by organisms, in vivo under the collective effect of various metabolic enzymes and fluid environment, chemical constitution changes, this
One process is referred to as being metabolized.Metabolic enzyme in hepatocyte microsome, mitochondria participates in the metabolic process of drug in vivo.And gene
Polymorphism, the difference of enzymatic activity and quantity can be caused, so as to cause individual difference the effect of drug, cause curative effect insufficient or poison
The generation of side effect.Therefore, drug therapy is caused the reason of huge individual difference occur, in addition to pathology traditionally, physiology, property
Not, age, height, weight, compliance etc. are outer, and the otherness of inherent cause is also a critically important reason.In nerve
During mental disease clinical application, the related gene of drug used is detected, personalized medicine scheme is provided, it can be most
Limits improve level of rational use of drugs, it is ensured that drug safety reduces the adverse reaction of drug, palliates the agonizing sufferings for patient as far as possible.
Currently, mainly having Sanger sequencings, quantitative fluorescent PCR and gene to the detection of psychoneural class medication related gene
Chip.Sanger sequencings, quantitative fluorescent PCR are since flux limitation is mainly for the common mutations site of a few gene.Base
Because though chip can realize high throughput, cost is high, flexibility is low, and depends on known gene pleiomorphism, is finding newly
There is limitation on mutational site, be unfavorable for the development of accumulation and the scientific research of data.The development of two generations sequencing makes to different
Body carries out genome sequencing and becomes a reality, and can excavate the hereditary variation of Different Individual DNA level comprehensively, but high cost is to restrict
Its widely applied bottleneck.Compared to above-mentioned technology, target area capture sequencing data accuracy higher, it is easier, economical,
Efficiently.The mutation of each known site of Different Individual can not only be illustrated, and improves the detectability to rare mutation.
Invention content
Clinical application in order to give psychoneural class drug provides guidance, and the present invention provides a kind of psychoneural class drug
The method and its application of related gene high-flux sequence, this method include the acquisition of genomic DNA, the expansion of target gene multiplex PCR
Increase reaction and product purification, joint sequence PCR reaction and product purification, library detection and high-flux sequence, we
Method can 29 SNP sites of disposable pair and relevant 20 genes of Nervous and mental diseases medication be detected and can cover anti-essence
The one two wires overwhelming majority such as refreshing Split disease, depression, antianxiety disease, anti-epileptic, parkinsonism, mostly dynamic obstacle, pain
Drug provides guidance for the medication of patient to greatest extent.
A kind of method of psychoneural class pharmaceutical relevant gene high-flux sequence, specifically comprises the following steps：
1) acquisition of genomic DNA：The extraction that mankind's complete genome DNA is carried out to human whole blood anti-freezing sample, after extraction
Carry out extraction and the purity detecting of DNA concentration；
2) reaction of target gene multiplexed PCR amplification and product purification：Human gene group DNA and multiple PCR primer, PCR are expanded
Increase reagent mixing, captures target gene DNA fragmentation；Using AgencourtAMPure XP magnetic beads to multiple after the completion of amplified reaction
PCR product is purified；
3) joint sequence PCR reactions and product purification：Multiple PCR products after purification are diluted 500-1000 times and are used as mould
Plate is added primer and PCR amplification reagent and carries out PCR reactions, after reaction using AgencourtAMPure XP magnetic beads to product into
Row purifying；
4) library detection：Using Agilient Bioanalyer 2100 and Q-PCR detection library concentrations and library fragments
Size；
5) high-flux sequence：High-flux sequence is carried out to library using Illumina microarray datasets.
In the method for psychoneural class pharmaceutical relevant gene high-flux sequence described above, the DNA concentration carries
It takes and is with method for detecting purity：The genomic DNA of acquisition is carried out using 2000 ultramicron ultraviolet specrophotometers of NanoDrop
It detects, between A260/A280=1.6-2.0, Qubit quantifies sample concentration C >=6ng/ μ l；DNA total amounts M >=250ng of extraction.
It is multiple in the step 2) in the method for psychoneural class pharmaceutical relevant gene high-flux sequence described above
PCR reactions carry out in same reaction tube, and the content of the human gene group DNA in reaction tube is 20-100ng.
The multi-PRC reaction program is：95 DEG C of pre-degeneration 5min, 1 cycle；95 DEG C of denaturation 30sec → 58 DEG C annealing
90sec → 72 DEG C extend 60sec, total 30-40 cycles；72 DEG C thoroughly extend 10min, 1 cycle；The multi-PRC reaction body
System includes 25 μ l, Multiplex of 11.5 μ l, Template DNA2 μ l, 2 × Multiplex Buffer of Primer Mix
DNA Polymerase 1 μ l, H2O 20.5μl。
PCR response procedures are in the step 3)：94 DEG C of pre-degeneration 2min, 1 cycle；94 DEG C of denaturation 15sec, 68 DEG C downward
60sec is stretched, 30-40 cycles are carried out；PCR reaction systems include 10 × Buffer for KOD-Plus 5 μ l, 2mM in step 3)
23 μ l, Template DNA of dNTPs 5 μ l, 25mM MgSO4,2 μ l, Primer Mix, 2 μ l, KOD-Plus- (1U/ μ l) 1 μ
l；The initial amount of DNA is 10-80ng/ reactions, a concentration of 0.02 μM -0.15 μM in system of primer in the step 3).
Purifying is carried out to PCR product using AgencourtAMPure XP magnetic beads and specifically includes following step：
(1) merge 50 μ l of moisturizing in product to 50 μ l PCR, reach 100 μ l of total volume, after equilibrium at room temperature is added
AMPure XP magnetic beads beat mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipes are placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and magnetic bead is added into pipe, and mixing is beaten for several times with pipettor suction；
(4) after being incubated at room temperature 5min, PCR pipe is placed in 5min on magnetic frame；
(5) supernatant is removed, PCR pipe continues to be placed on magnetic frame, be washed 2 times with 200 μ l, 80% ethanol solutions；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, residual ethanol is made thoroughly to wave
Hair；
(7) EP pipes are removed from magnetic frame, 30 μ l Nuclease-free water is added, pipettor, which is gently inhaled, plays resuspension
Magnetic bead avoids generating bubble, is stored at room temperature 5min；
(8) PCR pipe is replaced on magnetic frame, stands 3min；
(9) use pipettor Aspirate supernatant to get PCR product after purification；
The volume that AMPure XP magnetic beads are wherein added in step (1) in the purifying of step 2) multiple PCR products is 70 μ l-
80 μ l, the middle volume that AMPure XP magnetic beads are added of step (3) is 20 μ l-30 μ l；In the purifying of step 3) joint sequence PCR product
The volume that AMPure XP magnetic beads are added in step (1) is 50 μ l-60 μ l, and the volume of AMPure XP magnetic beads is added in step (3)
For 40 μ l-50 μ l.
The method of the present invention is used for the detection of patients' neural's antipsychotics related gene, and associated gene includes：MC4R、
HTR1A、DRD2、CYP2D6、ANKK1、HTR2C、CYP1A2、CYP3A5、NAT2、UGT2B15、CYP2C19、FKBP5、HTR2A、
CYP2C9, HLA-B, HLA-A, UGT1A6, CPS1, OPRM1, DRD3 totally 20 genes.It is specifically included：Genomic DNA obtains
It takes, target gene multiplexed PCR amplification reacts and product purification, joint sequence PCR reactions and product purification, library detection and height
Flux sequencing and etc..The method disclosed in the present patent application can be detected 29 mutational sites in above-mentioned 20 genes,
Testing result can provide medication guide for psychoneural class drug.It is specific as shown in table 1：
1 the method for the invention of table detects gene and corresponding mutational site
Corresponding multiple PCR primer includes 29 pairs of primers, specially：
5′-CCTACACGACGCTCTTCCGATCT-F-3′
5′-CAGACGTGTGCTCTTCCGATCT-R-3′
- F and-the R sequence in wherein each site are as follows：
Advantageous effect of the present invention compared with conventional art is：
(1) this method can disposable pair with 29 SNP sites of relevant 20 genes of Nervous and mental diseases medication into
Row detection can cover antipsychotic, depression, antianxiety disease, anti-epileptic, parkinsonism, mostly dynamic obstacle, pain
Deng a two wires overwhelming majority drug, extensive guide is provided for the medication of patient to greatest extent, broad covered area.
(2) this method to MC4R, HTR1A, DRD2, CYP2D6, ANKK1, HTR2C, CYP1A2, CYP3A5, NAT2,
UGT2B15, CYP2C19, FKBP5, HTR2A, CYP2C9, HLA-B, HLA-A, UGT1A6, CPS1, OPRM1, DRD3 totally 20 bases
29 mutational sites of cause carry out the sequencing of two generations after carrying out specific multiplexed PCR amplification by using primer disclosed in the present application
Method, substantially increase the detection sensitivity of psychoneural class pharmaceutical relevant gene mutation, while sample requirement amount is few, can be same
When detect multiple sites and be more advantageous to scientific research and Clinical practice.
(3) for this method compared with traditional Sanger PCR sequencing PCRs, flux is high, once can carry out target complete to multiple samples
The detection of gene mutation site, and accuracy is also suitable with Sanger sequencing approaches.Sequence, target are resurveyed compared to full-length genome
Areas captured sequencing amount wants low so that it be sequenced cost decline and also it is with strong points, coverage is deeper, data accuracy higher.
Therefore, screening and application of the capture sequencing in target area particularly suitable for large sample size, can not only verify mental disease gene
The variation of known site, and can be also found that genetic mutation rare/rare and that effect is strong.
Specific implementation mode
The present invention is further described below by way of specific embodiment, but the embodiment does not limit this hair in any way
The range of bright patent protection.
Embodiment 1：Psychoneural class pharmaceutical relevant gene is detected based on Illumina microarray datasets
Reagent：Multiplex PCR Kit (Vazyme), KOD-Plus-0902 (TOYOBO), TIANamp Blood
DNA Kit(TIANGEN)
1. the acquisition of genomic DNA：
Take three patients in psychiatric department：The anticoagulation sample of Sample1, Sample2, Sample3 are carried using Tiangeng blood DNA
Kit is taken, is extracted according to kit specification operating procedure.The genomic DNA of acquisition uses 2000 ultramicron of NanoDrop
Ultraviolet specrophotometer is detected A260/A280, and Qubit quantifies sample concentration C (ng/ μ l).As a result as follows：
| Sample | Sample1 | Sample2 | Sample3 |
| A260/A280 | 1.82 | 1.90 | 1.86 |
| C(ng/μl) | 31 | 29 | 38 |
2. first round multi-PRC reaction
Human gene group DNA is mixed with PCR primer, PCR amplification reagent, PCR reactions is carried out, target gene is expanded
Increase.System carries out in a reaction tube, without being in charge of.
(1) initial amount of DNA is 20-100ng/ reaction tubes；
(2) 0.02 μM -0.15 μM of the final concentration of every primer of primer.
2.1 reaction systems are as follows：
2.2 coded programs are as follows：
2.3 first round primers：
5′-CCTACACGACGCTCTTCCGATCT-F-3′
5′-CAGACGTGTGCTCTTCCGATCT-R-3′
- F and-the R sequence in wherein each site are as follows：
3. first round PCR product magnetic beads for purifying：Using AgencourtAMPure XP magnetic beads, to first round PCR product into
Row purifying.
(1) merge 50 μ l of moisturizing in product to 50 μ l PCR, reach 100 μ l of total volume, after 70 μ l equilibrium at room temperature are added
AMPure XP magnetic beads beat mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipes are placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and 20 μ l magnetic beads are added into pipe, and mixing is beaten for several times with pipettor suction；
(4) after being incubated at room temperature 5min, PCR pipe is placed in 5min on magnetic frame；
(5) supernatant is removed, PCR pipe continues to be placed on magnetic frame, be washed 2 times with 200 μ l, 80% ethanol solutions；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, residual ethanol is made thoroughly to wave
Hair；
(7) EP pipes are removed from magnetic frame, 30 μ l Nuclease-free water is added, pipettor, which is gently inhaled, plays resuspension
Magnetic bead avoids generating bubble, is stored at room temperature 5min；
(8) PCR pipe is replaced on magnetic frame, stands 3min；
(9) use pipettor Aspirate supernatant to get first round multiplex PCR product after purification.
4. the second wheel joint sequence PCR reactions
(1) first round multiple PCR products after purification are diluted 500-1000 times and is used as masterplate, the second wheel connector sequence is added
The initial amount of row primer and PCR reaction systems, DNA is 10-80ng/ reactions.
(2) 0.2 μM -0.4 μM of the final concentration of every primer of primer.
4.1 reaction systems are as follows：
4.2 response procedures are as follows
4.3 second wheel joint sequences：
5`-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3`
5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
-3
BarcodeX：NNNNNNNN
The BarcodeX of 3 patients in psychiatric department Sample1, Sample2, Sample3 is respectively：Barcode1、
Barcode2、Barcode3
Barcode1：AAGTCTCT
Barcode2：CCAGCGCT
Barcode3：ATGAACCT
5. the second wheel magnetic beads for purifying
Using AgencourtAMPure XP magnetic beads, the second wheel PCR product is purified.
(1) merge 50 μ l of moisturizing in product to 50 μ l PCR, reach 100 μ l of total volume, after 50 μ l equilibrium at room temperature are added
AMPure XP magnetic beads beat mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipes are placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and 40 μ l magnetic beads are added into pipe, and mixing is beaten for several times with pipettor suction；
(4) after being incubated at room temperature 5min, PCR pipe is placed in 5min on magnetic frame；
(5) supernatant is removed, PCR pipe continues to be placed on magnetic frame, be washed 2 times with 200 μ l, 80% ethanol solutions；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, residual ethanol is made thoroughly to wave
Hair；
(7) EP pipes are removed from magnetic frame, 30 μ l Nuclease-free water is added, pipettor, which is gently inhaled, plays resuspension
Magnetic bead avoids generating bubble, is stored at room temperature 5min；
(8) PCR pipe is replaced on magnetic frame, stands 3min；
(9) use pipettor Aspirate supernatant to get the second wheel PCR product after purification.
6. library detection
Using Agilient Bioanalyer2100 and Q-PCR detection library concentration and clip size.
7. high-flux sequence
It is detected using Illumina microarray datasets.
Embodiment 2
Take three patients in psychiatric department：The anticoagulation sample of Sample1, Sample2, Sample3 are detected, operating procedure
It is operated by embodiment one, following result is obtained after Illumina microarray datasets are sequenced and analyze：
(note：W indicates that wild type, M indicate that homozygous mutant, H indicate heterozygous mutant.The mankind belong to amphiploid biology, have
Two homologues.Wild type indicates not to be mutated on this two this sites of homologue；Homozygous mutant indicates
This site mutates on two chromosomes；Heterozygous mutant then indicates that wherein item chromosome is mutated, another
Chromosome does not mutate.)
For three patients, if attending physician intends to use drug Risperidone, and Risperidone drug metabolism mainly with
CYP2D6 enzymes are related, are normal wild type according to result above Sample1 CYP2D6 genotype, are eubolism class,
Enzymatic activity is normal；Sample2 carries * 2 and * 4 allelotypes, is intermediate supersession type, and enzymatic activity weakens；Sample3 carries *
4 and * 10 allelotypes, are intermediate supersession type, and enzymatic activity weakens.Doctor can refer to result above adjustment patient and use medicament
Amount carries out precision medication to patient.
Embodiment 3
Take three patients in psychiatric department：The anticoagulation sample of Sample1, Sample2, Sample3 are detected, and are used
Sanger methods are sequenced, and sequencing result is compared with patent of the present invention, as a result such as following table：
As seen from the above table, this method and the Sanger PCR sequencing PCR acquired results that goldstandard is sequenced by positioning are completely the same.But
Sanger PCR sequencing PCR detection sites are few, and detection time is long；Sequence is resurveyed compared to full-length genome, this method (target area capture) is surveyed
Sequence amount wants low so that it be sequenced cost decline and also it is with strong points, coverage is deeper, data accuracy higher.
Claims (9)
1. a kind of method of psychoneural class pharmaceutical relevant gene high-flux sequence, specifically comprises the following steps：
1) acquisition of genomic DNA：The extraction that mankind's complete genome DNA is carried out to human whole blood anti-freezing sample, carries out after extraction
The extraction of DNA concentration and purity detecting；
2) reaction of target gene multiplexed PCR amplification and product purification：Human gene group DNA and multiple PCR primer, PCR amplification are tried
Agent mixes, and captures target gene DNA fragmentation；Using AgencourtAMPure XP magnetic beads to multiplex PCR after the completion of amplified reaction
Product is purified；
3) joint sequence PCR reactions and product purification：Multiple PCR products after purification are diluted 500-1000 times and are used as template,
Primer is added and PCR amplification reagent carries out PCR reactions, it is pure to product progress using AgencourtAMPure XP magnetic beads after reaction
Change；
4) library detection：Using Agilient Bioanalyer2100 and Q-PCR detection library concentration and library fragments size；
5) high-flux sequence：High-flux sequence is carried out to library using Illumina microarray datasets.
2. the high-flux sequence method of the psychoneural class pharmaceutical relevant gene according to claim l, which is characterized in that institute
The extraction for the DNA concentration stated and method for detecting purity are：The genomic DNA of acquisition is purple using 2000 ultramicron of NanoDrop
Outer spectrophotometer is detected, and between A260/A280=1.6-2.0, Qubit quantifies sample concentration C >=6ng/ μ l；Extraction
DNA total amounts M >=250ng.
3. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, which is characterized in that institute
The multiple PCR primer stated includes 29 pairs of primers, specially：
5′-CCTACACGACGCTCTTCCGATCT-F-3′
5′-CAGACGTGTGCTCTTCCGATCT-R-3′
- F and-the R sequence in wherein each site are as follows：
4. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, it is characterised in that：Institute
It states multi-PRC reaction in step 2) to carry out in same reaction tube, the content of the human gene group DNA in reaction tube is 20-
100ng；The multi-PRC reaction program is：95 DEG C of pre-degeneration 5min, 1 cycle；95 DEG C of denaturation 30sec → 58 DEG C annealing
90sec → 72 DEG C extend 60sec, total 30-40 cycles；72 DEG C thoroughly extend 10min, 1 cycle；The multi-PRC reaction body
System includes 25 μ l, Multiplex of 11.5 μ l, Template DNA of Primer Mix, 2 μ l, 2 × Multiplex Buffer
DNA Polymerase1 μ l, H2O20.5μl。
5. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, which is characterized in that step
It is rapid 3) in PCR response procedures be：94 DEG C of pre-degeneration 2min, 1 cycle；94 DEG C are denaturalized 15sec, extend 60sec at 68 DEG C, carry out
30-40 is recycled；PCR reaction systems include 10 × Buffer for KOD-Plus, 5 μ l, 2mM dNTPs, 5 μ l in step 3),
2 μ l, Primer Mix of 25mM MgSO4,23 μ l, Template DNA, 21 μ l of μ l, KOD-Plus- (1U/ μ l)；The step
3) initial amount of DNA is 10-80ng/ reactions, a concentration of 0.02 μM -0.15 μM in system of primer in.
6. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, which is characterized in that institute
It includes joint sequence, upstream sequence 5`- to state PCR reactions in step 3)
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT-3 ｀；Downstream sequence is 5 '-
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCT CTTCCGATCT-3 ｀.
7. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, which is characterized in that adopt
Purifying is carried out to PCR product with AgencourtAMPure XP magnetic beads and specifically includes following step：
(1) merge 50 μ l of moisturizing in product to 50 μ l PCR, reach 100 μ l of total volume, the AMPure XP after equilibrium at room temperature are added
Magnetic bead beats mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipes are placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and magnetic bead is added into pipe, and mixing is beaten for several times with pipettor suction；
(4) after being incubated at room temperature 5min, PCR pipe is placed in 5min on magnetic frame；
(5) supernatant is removed, PCR pipe continues to be placed on magnetic frame, be washed 2 times with 200 μ l, 80% ethanol solutions；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, residual ethanol is made thoroughly to volatilize；
(7) EP pipes are removed from magnetic frame, 30 μ l Nuclease-free water is added, pipettor, which is gently inhaled to beat, is resuspended magnetic
Pearl avoids generating bubble, is stored at room temperature 5min；
(8) PCR pipe is replaced on magnetic frame, stands 3min；
(9) use pipettor Aspirate supernatant to get PCR product after purification；
The volume that AMPure XP magnetic beads are wherein added in step (1) in the purifying of step 2) multiple PCR products is 70 μ l-80 μ l,
The volume that AMPure XP magnetic beads are added in step (3) is 20 μ l-30 μ l；Step in the purifying of step 3) joint sequence PCR product
(1) volume that AMPure XP magnetic beads are added in is 50 μ l-60 μ l, and the volume that AMPure XP magnetic beads are added in step (3) is 40 μ
l-50μl。
8. the high-flux sequence method of psychoneural class pharmaceutical relevant gene according to claim 1, which is characterized in that its
29 SNP mutation sites of totally 20 genes can be sequenced, wherein 20 genes include：MC4R、HTR1A、
DRD2、CYP2D6、ANKK1、HTR2C、CYP1A2、CYP3A5、NAT2、UGT2B15、CYP2C19、FKBP5、HTR2A、
CYP2C9, HLA-B, HLA-A, UGT1A6, CPS1, OPRM1 and DRD3.
9. the high-flux sequence method of the psychoneural class pharmaceutical relevant gene described in claim 1-8 is in psychoneural class drug
Clinical application guidance in application, the psychoneural class drug include antipsychotic, depression, antianxiety disease,
Anti-epileptic, parkinsonism, mostly dynamic obstacle, pain therapy drug.
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