CN108342466B - 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, the reaction of target gene multiplexed PCR amplification and product purification, joint sequence PCR reaction and product purification, library detection and high-flux sequence and etc., this method can 29 SNP sites disposably to 20 genes relevant to Nervous and mental diseases medication detect and can cover antipsychotic, depression, antianxiety disease, anti-epileptic, parkinsonism, mostly dynamic obstacle, the one two wires overwhelming majority drug such as pain, guidance is provided to greatest extent 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 technique
Currently, great change has occurred in human diseases spectrum, and neuropsychiatric disease is more and more forward in human diseases pedigree,
And its disease incidence is also constantly being increased with the increasingly increased work and life pressure that people are faced.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 are 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
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 treatment method of preceding overwhelming majority mental disease and means.Mental disease generally requires the continued treatment under drug control,
But there is potential adverse 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, causes to patient
Significant impact.
Antipsychotics mechanism of action, which 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. to the blocking of histamine receptor, blocking mainly to H1 receptor can produce calm and weight
Increased adverse reaction；2. the blocking to adrenergic receptor can produce sedation and orthostatic mainly to 1 receptor of α
The adverse reaction of low blood pressure, tachycardia, hypogona dism；3. to the blocking effect of cholinergic recepter, mainly to M1 receptor
Blocking, can produce the adverse reactions such as dry, constipation, intestinal obstruction, dysuria；4. the blocking to dopamine receptor, can produce
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 structure changes, this
One process, which is referred to as, to be 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 the curative effect individual difference 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 as far as possible, palliates the agonizing sufferings for patient.
Currently, mainly having Sanger sequencing, quantitative fluorescent PCR and gene to the detection of psychoneural class medication related gene
Chip.Sanger sequencing, quantitative fluorescent PCR are since flux limitation is mainly for the common mutations site of a few gene.Base
Because though chip is able to achieve high throughput, cost is high, flexibility is low, and depends on known gene pleiomorphism, new in discovery
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 is so as 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 it is 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.
Summary of the invention
Guidance is provided in order to give the clinical application of psychoneural class drug, 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 disposably to 20 genes relevant to Nervous and mental diseases medication detect 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 to greatest extent for the medication of patient.
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 of mankind's complete genome DNA is carried out to the anticoagulant sample of human whole blood, after extraction
Carry out the extraction and 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；Use AgencourtAMPure XP magnetic bead to multiple after the completion of amplified reaction
PCR product is purified；
3) joint sequence PCR reaction 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 reaction, after reaction using AgencourtAMPure XP magnetic bead to product into
Row purifying；
4) library detection: using Agilient Bioanalyer 2100 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 dataset.
In the method for psychoneural class pharmaceutical relevant gene high-flux sequence described above, the DNA concentration is mentioned
It takes and method for detecting purity are as follows: use 2000 ultramicron ultraviolet specrophotometer of NanoDrop to carry out the genomic DNA of acquisition
It detects, between A260/A280=1.6-2.0, Qubit quantifies sample concentration C >=6ng/ μ l；DNA total amount 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 reaction carries 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 are as follows: 95 DEG C of initial denaturation 5min, 1 circulation；95 DEG C of denaturation 30sec → 58 DEG C annealing
90sec → 72 DEG C extend 60sec, total 30-40 circulation；72 DEG C thoroughly extend 10min, 1 circulation；The multi-PRC reaction body
System includes 11.5 μ l, Template DNA2 μ l, 2 × Multiplex Buffer of Primer Mix 25 μ l, Multiplex
DNA Polymerase 1 μ l, H2O 20.5μl。
PCR response procedures in the step 3) are as follows: 94 DEG C of initial denaturation 2min, 1 circulation；94 DEG C of denaturation 15sec, 68 DEG C downward
60sec is stretched, 30-40 circulation is carried out；PCR reaction system includes 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/ reaction in the step 3), and concentration of the primer in system is 0.02 μM -0.15 μM.
PCR product is purified using AgencourtAMPure XP magnetic bead specifically include the following steps:
(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 bead plays mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipe is placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and magnetic bead is added into pipe, plays mixing 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 solution；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, wave residual ethanol thoroughly
Hair；
(7) EP pipe is 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) with pipettor Aspirate supernatant to get PCR product after purification；
The volume that AMPure XP magnetic bead is 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 bead is 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 bead is added in step (1) is 50 μ l-60 μ l, and the volume of AMPure XP magnetic bead is added in step (3)
For 40 μ l-50 μ l.
Method of the invention be used for patients' neural's antipsychotics related gene detection, associated gene include: 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 reaction and product purification, library detection and height
Flux sequencing and etc..The method disclosed in the present patent application can detect 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, specifically:
5′-CCTACACGACGCTCTTCCGATCT-F-3′
5′-CAGACGTGTGCTCTTCCGATCT-R-3′
Wherein-F and-the R sequence in each site are as follows:
Beneficial effect of the present invention compared with conventional art is:
(1) this method can disposably 29 SNP sites to 20 genes relevant to 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 use.
(3) for this method compared with traditional Sanger PCR sequencing PCR, 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 approach.Sequence, target are resurveyed compared to full-length genome
Areas captured sequencing amount wants low so that its sequencing cost decline and also it is with strong points, coverage is deeper, data accuracy is 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 embodiment
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 dataset
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 mentioned 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 carries out detection 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 reaction 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 tube；
(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′
Wherein-F and-the R sequence in each site are as follows:
3. first round PCR product magnetic beads for purifying: use AgencourtAMPure XP magnetic bead, 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 bead plays mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipe is placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and 20 μ l magnetic beads are added into pipe, plays mixing 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 solution；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, wave residual ethanol thoroughly
Hair；
(7) EP pipe is 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) with pipettor Aspirate supernatant to get first round multiplex PCR product after purification.
4. the second wheel joint sequence PCR reaction
(1) first round multiple PCR products after purification are diluted 500-1000 times and is used as template, the second wheel connector sequence is added
Column primer and PCR reaction system, the initial amount of DNA are 10-80ng/ reaction.
(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 be respectively as follows: Barcode1,
Barcode2、Barcode3
Barcode1:AAGTCTCT
Barcode2:CCAGCGCT
Barcode3:ATGAACCT
5. the second wheel magnetic beads for purifying
Using AgencourtAMPure XP magnetic bead, 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 bead plays mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipe is placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and 40 μ l magnetic beads are added into pipe, plays mixing 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 solution；
(6) it removes remaining ethanol solution as far as possible with pipettor, is stored at room temperature 3-5min, wave residual ethanol thoroughly
Hair；
(7) EP pipe is 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) with pipettor Aspirate supernatant to get the second wheel PCR product after purification.
6. library detection
Library concentration and clip size are detected using Agilient Bioanalyer2100 and Q-PCR.
7. high-flux sequence
It is detected using Illumina microarray dataset.
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 dataset is sequenced and analyzes:
(note: W indicates wild type, and M indicates 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
There is no mutation for chromosome.)
For three patients, if attending physician intends to use drug Risperidone, and Risperidone drug metabolism mainly with
CYP2D6 enzyme is related, is normal wild type according to result above Sample1 CYP2D6 genotype, is 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
The sequencing of Sanger method, sequencing result are compared with the invention patent, the result is as follows:
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 site is 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 its sequencing cost decline and also it is with strong points, coverage is deeper, data accuracy is higher.
Claims (8)
1. multiple PCR primer is applied in preparing psychoneural class pharmaceutical relevant gene high-flux sequence reagent, it is characterised in that:
The psychoneural class pharmaceutical relevant gene high-flux sequence includes the following steps:
1) acquisition of genomic DNA: the extraction of mankind's complete genome DNA is carried out to the anticoagulant sample of human whole blood, is carried out after extraction
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 mixing, captures target gene DNA fragmentation；Use AgencourtAMPure XP magnetic bead to multiplex PCR after the completion of amplified reaction
Product is purified；
3) joint sequence PCR reaction and product purification: diluting 500-1000 times for multiple PCR products after purification and be used as template,
Primer is added and PCR amplification reagent carries out PCR reaction, it is pure to product progress using AgencourtAMPure XP magnetic bead 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 dataset；
The multiple PCR primer includes 29 pairs of primers, specifically:
5′-CCTACACGACGCTCTTCCGATCT-F-3′
5′-CAGACGTGTGCTCTTCCGATCT-R-3′
Wherein the upstream primer and downstream primer sequence in each site are as follows:
Serial number upstream primer-F downstream primer-R
1 GGCAATCTTCTGTTCTGAGACAATA TCTGCTGAAACTGTGCTTGGTAT
2 CTGAAGGCTGATGGTTGGAGTT TTTTCAGTAGGAGCTTTTAGAAACAAC
3 CCCTGCTTTCGGCTGCG GTCCACGCCCGCAACAAG
4 TGGGAAGGCGGGGGACGGG TGCTCACGGCTTTGTCCAAGAGA
5 GGACCTGATGCACCGGCG TTCTGGAAGTCCACATGCAGCAG
6 GCGTGAGCCCATCTGGGAAA GGTCAGGCTTACAGGATCCTGGT
7 GTGGTCACCATCCCGGCAGA TACCCCGTTCTGTCCCGAGTATG
8 CCCGGCCCAGCCACCATG GGGGCACAGCACAAAGCTCA
9 CCGCTTTGTGCCCTTCTG TTCATGGCCACGCGCAC
10 TAGCCGCTCCAAAGGCAGTA CACGTAATGCTGAGTGCTGATTG
11 CAGAATCTTGCTCTGTCACCCA ATATGTCAGCTGGGACCGGT
12 TCATGTTGGGAATCTTGAGGCT TGATAAACACTGATGCGTGTTCTG
13 TGCTCTACTGTCATTTCTAACCATAATC CACACAGGAGCCACCCAAGG
14 GGGTATTTTTACATCCCTCCAGTTA ATTCCTCTCTCTTCTGTCAAGCAG
15 GGACCAAATCAGGAGAGAGCAGT AATGATGTGGTTATAAATGAAGATGTTG
16 TCAATGCCAGTAAATCATCTGCTAT TTACTGTAGTCATAATATTCCCAACACA
17 TCTTAGATATGCAATAATTTTCCCA AAAATATCACTTTCCATAAAAGCAAG
18 TGGAAAAATTGAATGAAAACATCAG GGATTTCCCAGAAAAAAAGACTG
19 GGAATGTTGTATTTTTATCTGGCAA GGGTTTGTTGTAGAGATTATTTAATCATT
20 GGAAAGAACGCTGAGTTGATGTAAT GATGTCATTTATCTCCACCTTCCA
21 GTTTCTCTTCCTGTTAGGAATTGTTT GTCCAGTAAGGTCAGTGATATGGAG
22 GAACGTGTGATTGGCAGAAACC GGGAATGAGATAGTTTCTGAATTTAAT
23 GAGGAGGCTACAGCAATAATCATGA CAAGAGTTGGAACCATGGTTATGC
24 AAAAAGGAGCAGGTGCATGAAG ACTAAGGTTATCGCAGCAACAGT
25 TCACAGCTTGTAAAGGTGAGAGC CCCACCTCCTCACATTATGCC
26 TCACAGACCCAGCCTTACCC GCCACTCGTTGGGAAAAAG
27 CAGCTGTTTGCCACGGAAG TTCAGAAAACAGTATGCCTAGTTCTTAC
28 CACTGATGCCTTGGCGTACTC ATGGAGGGACTGCCGGTC
29 AGTAGGCATGTGGGCGGG GGAAGCCCCTTGGCATCA。
2. multiple PCR primer is preparing psychoneural class pharmaceutical relevant gene high-flux sequence reagent according to claim 1
Middle application, it is characterised in that: the DNA concentration and method for detecting purity are as follows: use the genomic DNA of acquisition
NanoDrop2000 ultramicron ultraviolet specrophotometer is detected, and between A260/A280=1.6-2.0, Qubit quantifies sample
Concentration C >=6ng/ μ l；DNA total amount M >=250ng of extraction.
3. multiple PCR primer according to claim 1 is preparing the examination of psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in agent, it is characterised in that: multi-PRC reaction carries out in same reaction tube in the step 2), people's base in reaction tube
Because the content of group DNA is 20-100ng；The multi-PRC reaction program are as follows: 95 DEG C of initial denaturation 5min, 1 circulation；95 DEG C of denaturation
Anneal 90sec → 72 DEG C extension 60sec for 30sec → 58 DEG C, total 30-40 circulation；72 DEG C thoroughly extend 10min, 1 circulation；It is described
Multi-PRC reaction system include 11.5 μ l of primer mixture (Primer Mix), template DNA (Template DNA) 2 μ l, 2
25 μ l of × multi-buffer liquid (Multiplex Buffer), 1 μ l of multiple dna polymerase (MultiplexDNA Polymerase),
H2O 20.5μl。
4. multiple PCR primer according to claim 1 is preparing the examination of psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in agent, which is characterized in that PCR response procedures in step 3) are as follows: 94 DEG C of initial denaturation 2min, 1 circulation；94 DEG C of denaturation 15sec,
Extend 60sec at 68 DEG C, carries out 30-40 circulation；PCR reaction system includes 10 × KOD-Plus buffer, 5 μ l in step 3),
5 μ l, 25mM MgSO of 2mM dNTPs42 μ l, 23 μ l of primer mixture (Primer Mix), template DNA (Template DNA) 2
The 1 μ l of KOD-Plus of μ l, 1U/ μ l；The initial amount of DNA is 10-80ng/ reaction in the step 3), and primer is in system
Concentration is 0.02 μM -0.15 μM.
5. multiple PCR primer according to claim 1 is preparing the examination of psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in agent, which is characterized in that including joint sequence, upstream sequence 5`- in PCR reaction in the step 3)
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT CT-3 `；
Downstream sequence is 5 '
- CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCT CTTCCGATCT-3 `；
Wherein NNNNNNNN is bar code X (BarcodeX).
6. multiple PCR primer according to claim 1 is preparing the examination of psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in agent, which is characterized in that purifying is carried out to PCR product using AgencourtAMPure XP magnetic bead and specifically includes following steps
It is rapid:
(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 is added
Magnetic bead plays mixing for several times with pipettor suction；
(2) after being incubated at room temperature 5min, EP pipe is placed in 5min on magnetic frame；
(3) transfer supernatant is managed to new EP, and magnetic bead is added into pipe, plays mixing 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 solution；
(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 pipe is removed from magnetic frame, the water (Nuclease-free water) of 30 μ l nuclease frees is added, pipettor is light
Light inhale beats resuspension magnetic bead, avoids generating bubble, is stored at room temperature 5min；
(8) PCR pipe is replaced on magnetic frame, stands 3min；
(9) with pipettor Aspirate supernatant to get PCR product after purification；
The volume that AMPure XP magnetic bead is 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 bead is 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 bead is added in is 50 μ l-60 μ l, and the volume that AMPure XP magnetic bead is added in step (3) is 40 μ
l-50μl。
7. multiple PCR primer according to claim 1 is preparing the examination of psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in agent, which is characterized in that 29 SNP mutation sites of totally 20 genes can be sequenced in it, wherein described 20
Gene include: MC4R, HTR1A, DRD2, CYP2D6, ANKK1, HTR2C, CYP1A2, CYP3A5, NAT2, UGT2B15,
CYP2C19, FKBP5, HTR2A, CYP2C9, HLA-B, HLA-A, UGT1A6, CPS1, OPRM1 and DRD3.
8. multiple PCR primer as claimed in claim 1 to 7 is preparing psychoneural class pharmaceutical relevant gene high-flux sequence
It is applied in reagent, the psychoneural class drug includes antipsychotic, depression, antianxiety disease, anti-epileptic, pa gold
Gloomy syndrome, mostly dynamic obstacle, pain therapy drug.
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