CN104928375B - Method that is a kind of while measuring people's cyclosporine action target spot gene pleiomorphism - Google Patents
Method that is a kind of while measuring people's cyclosporine action target spot gene pleiomorphism Download PDFInfo
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Abstract
Method that is provided by the invention a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, it is i.e. a kind of to measure the cyclosporine multiple single nucleotide polymorphism of action target spot access series protein coding gene (single nucleotide polymorphism simultaneously, SNP) the method for loci gene type, it is easy to operate, without sequence-specific probes, one chip can carry out Multiple detection to 384 samples, each system at most can be achieved 40 and react again, 40 SNP sites are detected simultaneously, flux can be according to requiring to carry out personalized adjustment, it is high-throughput, it is applied widely, tens arrive thousands of a samples, hundreds and thousands of a sites are arrived in detection tens simultaneously；Without fluorescent marker, it is only necessary to synthesize general primer, single analysis cost is low；High flexibility ratio, on a chip sample and site primer matching can choose at random；Sample size needed for analysis is few (10ng), highly sensitive, and accuracy of detection is high.
Description
Technical field
The present invention relates to technical field of molecular biology more particularly to it is a kind of measure simultaneously cyclosporine (Cyclosporine,
CsA) the multiple single nucleotide polymorphism of action target spot access series protein coding gene (single nucleotide
Polymorphism, SNP) loci gene type method, i.e., it is a kind of while measure people's cyclosporine action target spot gene pleiomorphism
Method.
Background technology
Ciclosporin in treating window is narrow, and curative effect is there are notable individual difference, at present, clinically usually by carrying out medicine to it
Object monitors (therapeutic drug monitoring, TDM), that is, blood concentration is monitored, to adjust the dosage of cyclosporine.But
Traditional TDM is difficult to from the variation between the angle reflection individual of pharmacodynamics, and TDM can only be implemented after the tablet has been ingested, nothing
Method predicts reaction of the body to drug before medication, and the predose of drug is difficult to determine.Therefore, it is carried out by TDM merely
The individualized treatment of cyclosporine is clearly inadequate, and there is an urgent need to find new individualized treatment strategy.
It is one of main factor for causing to make a variation between drug effect individual prior art discloses hereditary variation.By grinding
Study carefully the relationship of hereditary variation and drug response, the predose of drug can be formulated according to genotype, it is anti-drug can also to be screened
Sensitive or invalid specific crowd is answered, carries out individualized treatment, pharmacogenomics are the new platforms of Individual drug treatment development
Rank.To in August, 2014, FDA has been approved by or revises 138 kinds of drug specifications, it is proposed that detection genotype, to improve drug use
Validity and safety.
Pharmacogenomics study the genetic polymorphism for being related to pharmacokinetics and pharmacodynamics related gene.Cyclosporine is in body at present
Interior action target spot and access is clear and definite.Cyclosporine enter after T cell with a kind of immunophilin-cyclophilin
(Cyclophilin, CyP) is combined, and CyP-CsA compounds can combine and inhibit calcineurin in endochylema in specific manner
(calcineurin, CaN), activity block activating T cell nuclear factor (nuclear factor of activated T
Cell, NFAT) dephosphorylation, and then inhibit IL-2 genetic transcription, so as to inhibit the subsequent activation of T cell.In cyclosporine
The functional status of the important molecule (CyP, CaN and NFAT) of actuating signal access, for the performance of cyclosporine pharmacological action, is played
Vital effect.The encoding gene mononucleotide polymorphic of CyP, CaN and NFAT in cyclosporine action target spot signal path
Property, influence gene function, and then influence the mutual of cyclosporine and CyP, CyP and CaN, CaN and NFAT and NFAT and target gene
With reference to so as to finally influence the immunosuppressive action of cyclosporine.
CyP belongs to a subclass of immunophilin, it has now been found that its encoding gene is PPIA.CaN is with heterodimer
Form exists, by the adjusting subunit Calcineurin B (CNB) of the catalytic subunit Calcineurin A (CNA) and 19ku of 61ku
Composition.There are three hypotype, CNA α, CNA β and CNA γ by CNA, and encoding gene is respectively PPP3CA, PPP3CB and PPP3CC.CNB
There are two hypotypes, are CNB1 and CNB2 respectively, encoding gene is PPP3R1 and PPP3R2 respectively.Wherein CNA α, CNA β and
CNB1 wide expressions in vivo, and CNA γ and CNB 2 is then existed only in brain and testis.NFAT is to belong to Rel albumen man
Race, Rel protein families include NF-kB and NFAT, are conservative DNA binding domain.NFAT has 5 kinds of hypotypes, be respectively NFATC1,
NFATC2、NFATC3、NFATC4、NFAT5.Wherein it is immune to be primarily present in T lymphocytes etc. by NFATC1, NFATC2, NFATC3
In cell, NFATC4 is present in other histocytes including heart.NFAT5, different from other 4 kinds of albumen, not by
The adjusting of intracellular calcium concentration mainly by activating tumor necrosis factor (TNF) genetic transcription of hypertonic induction, participates in T
Cell growth, the adjusting of development.CaN-NFAT signal paths have all played important work in immune system and non-immunological systems
With.The encoding gene of CyP, CaN and NFAT have single nucleotide polymorphism.In conclusion detection cyclosporine target spot pathway gene
Hereditary variation to prediction cyclosporine drug response have more important clinical meaning.
The performance of drug final effect in vivo, is influenced by multiple links, including pharmacokinetics and pharmacodynamics link.And
Pharmacokinetics and pharmacodynamics respectively receive the influence of a variety of enzymes, transport protein, receptor, signal path etc. again.And each receptor, albumen
Deng encoding gene, and there are multiple SNP sites.Therefore, a kind of genotype of a SNP site of gene is only detected, it is right
Explain the individual difference of drug, it is clear that be far from being enough.Therefore, it generally requires to detect the multiple SNP sites of multiple genes simultaneously.
At present, the detection method of gene pleiomorphism mainly has DNA direct sequencings and Taqman sonde methods etc..The DNA
Direct sequencing is the goldstandard of abrupt climatic change, but there are time-consuming and laborious, cost costly the defects of, be a kind of inspection of small throughput
Survey method is not suitable for being detected great amount of samples.The Taqman is the quantitative PCR technique of high special, its main feature is that
It is adapted to a small amount of SNP site detection of large sample, is a kind of SNP detections of moderate fluxes, and it is SNP multiple for detecting simultaneously
Point, then it is costly, it is not suitable for.
Invention content
To solve the above problem of the prior art, present invention offer is a kind of efficient, sensitive, accurate, can detect simultaneously
The method of multiple SNP sites, i.e., it is a kind of to measure the multiple SNP sites of cyclosporine action target spot access series protein coding gene simultaneously
The method of genotype is adapted to large sample or small sample, with further to assist cyclosporine individual administration, ensureing drug safety
Technical support is provided with effective.
In order to solve the above-mentioned technical problem, technical solution proposed by the present invention is：
Method that is a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, includes the following steps：
Step 1：The genomic DNA of peripheral blood cells sample is extracted, sieve is designed for the different mutational site of target gene
Select primer；And it is combined into different PCR reaction systems and carries out multi-PRC reaction；
Step 2：After the PCR product SAP processing of amplification, single base extension is carried out；
Step 3：After carrying out chip point sample, it is detected with Matrix-assisted laser desorption ionization；
Step 4：Testing result is analyzed using software, obtains typing data.
To optimize above-mentioned technical proposal, the measure that the present invention takes further includes：
Genomic DNA is extracted using DNA extraction kit in above-mentioned steps 1, and the DNA concentration of extraction is adjusted to 10-
100ng/ul。
Above-mentioned PCR response procedures are：94 DEG C of 15min, 45 cycles (94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min), 72
℃3min。
Above-mentioned SAP response procedures are：37 DEG C of 40min, 85 DEG C of 5min.
Above-mentioned single base extension condition is：94 DEG C of 30sec, 40 cycles [94 DEG C of 5sec, 5 (52 DEG C of partial circulatings
5sec, 80 DEG C of 5sec)], 72 DEG C of 3min.
Also there is the step of product purification, the operation of product purification is between above-mentioned steps 2 and step 3：
(1) resin uniform fold on resin scraper plate is taken, places 20min；
(2) the orifice plate 1000rpm that end is reacted in step 2 is centrifuged into 1min, 25 μ L deionized waters is added in per hole, are upside down in
It above resin plate, then inverts and resin plate is buckled on orifice plate, percussion makes resin fall into orifice plate, sealer；
(3) overturning orifice plate 20 minutes, 3500rpm, centrifugation 5min.
It is multiple in detection cyclosporine action target spot signal path while the method for the present invention can be efficient, sensitive, accurate
The genotype of multiple SNP sites in protein coding gene, the individual difference to explain clinical cyclosporine drug effect provide it is important according to
According to the clinical individualization of cyclosporine is instructed to be administered.
The present invention uses time-of-flight mass spectrometry (MALDI-TOF) method, and first passing through PCR amplification headed by technical principle contains
Then the genomic fragment of SNP realizes Single base extension, subsequent sample analytes and chip matrix by sequence specific primers
It is excited in vacuum tube by instantaneous nanosecond (10-9s) light laser after cocrystallization.Nucleic acid molecules therefore desorption become single charge from
Son, since the electric field intermediate ion flight time is inversely proportional with mass of ion, during by detecting flight of the nucleic acid molecules in vacuum tube
Between and obtain the accurate molecular weights of sample analytes, so as to detect SNP site information.Therefore, this method is suitable for examining simultaneously
Survey the multiple SNP sites of same sample.
The method of the invention is easy to operate, without sequence-specific probes, and a chip can carry out 384 samples more
Re-detection, each system at most can be achieved 40 to react again, while detect 40 SNP sites, and flux can be according to requiring to carry out individual character
Change adjustment, high-throughput, applied widely, tens arrive thousands of a samples, while detect tens to hundreds and thousands of a sites；Nothing
Need fluorescent marker, it is only necessary to synthesize general primer, single analysis cost is low；High flexibility ratio, sample and site primer on a chip
Matching can choose at random；Sample size needed for analysis is few (10ng), highly sensitive, and accuracy of detection is high, and SNP Detection accuracies are reachable
99.9%.It is suitble to routine clinical genetic test, further provides technical support for the clinical individual rational use of medicines.
Description of the drawings
Fig. 1 is the Genotyping figure of one reaction system of a sample；
Fig. 2 is rs6463247 sites CC type parting figures in embodiment 1；
Fig. 3 is rs6463247 sites CT type parting figures in embodiment 1；
Fig. 4 is rs6463247 sites TT type parting figures in embodiment 1；
Fig. 5 is rs7560138 sites AA type parting figures in embodiment 1；
Fig. 6 is rs7560138 sites AG type parting figures in embodiment 1；
Fig. 7 is rs7560138 sites GG type parting figures in embodiment 1.
Specific embodiment
The present invention provides method that is a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, including following step
Suddenly：
Step 1：The genomic DNA of peripheral blood cells sample is extracted, sieve is designed for the different mutational site of target gene
Select primer；And it is combined into different PCR reaction systems and carries out multi-PRC reaction；
Step 2：After the PCR product SAP processing of amplification, single base extension is carried out；
Step 3：After carrying out chip point sample, it is detected with Matrix-assisted laser desorption ionization；
Step 4：Testing result is analyzed using software, obtains typing data.
The side that is a kind of while measuring the multiple SNP site polymorphisms of cyclosporine action target spot access serial genes of the present invention
Method, i.e., a kind of while method that measures people's cyclosporine action target spot gene pleiomorphism, is realized especially by following steps：
1st, people's Whole Blood Genomic DNA is extracted
Whole Blood Genomic DNA is extracted, and adjust concentration and adjust a concentration of 10-100ng/ul with DNA extraction kit.
2nd, multi-PRC reaction
1) objective gene sequence is searched, for mutational site, designs and synthesizes PCR primer
2) according to sample 384 hole reaction tables of establishment have been extracted, the number per the corresponding DNA sample in hole, the primer are indicated.
3) 1 μ L DNA profilings are separately added into each hole of 384 orifice plates by table, pad pasting is spare after centrifugation in 2000rpm/10 seconds.
4) according to the form below prepares PCR reaction solution：(by taking 384 samples as an example)
Note：More than 384 hole reaction solutions have it is 4% excessive.
5) 12 union of a row is taken, the PCR reaction solution 133 μ L being configured are added in per hole, it is spare after of short duration centrifugation.
6) PCR reaction solution 4ul in 12 unions is taken with the 10ul volley of rifle fires, adds in 384 orifice plates for having 1uL DNA, make each hole
Final volume is 5ul.
7) PCR instrument will be put into after the of short duration centrifugation of 384 orifice plates equipped with 5ul reaction solutions, runs the reaction interval of entitled " PCR "
Sequence.PCR response procedures are：94 DEG C of 15min, 45 cycles (94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min), 72 DEG C of 3min.
8), will be spare after the of short duration centrifugation of 384 orifice plates after PCR response procedures, it can be in 4 DEG C of preservations.
3rd, SAP processing
1) SAP reaction solutions are prepared
SAP reaction solutions are prepared by following table order (by taking 384 samples as an example)
Note：More than 384 hole reaction solutions have it is 4% excessive.
2) 12 union of a row is taken, SAP reaction solutions with every 66 μ L of hole are dispensed, after of short duration centrifugation, are sub-packed in the 10ul volley of rifle fires
384 hole PCR reaction plates add 2 μ L, sealer, centrifugation per hole.
3) 384 orifice plates for having added in SAP reaction solutions are put into PCR instrument, run the response procedures of entitled " SAP ".SAP is anti-
Answer program：37 DEG C of 40min, 85 DEG C of 5min.
4) reaction terminates, and the of short duration centrifugation of 384 orifice plates taking-up is spare, can be in 4 DEG C of preservations.
4th, extension
1) iPlex reaction reagents are prepared
Note：More than 384 hole reaction solutions have it is 4% excessive.
2) 12 union of a row is taken, iPlex reaction solutions are dispensed with every 66 μ L of hole, after of short duration centrifugation, is dispensed with the 10uL volley of rifle fires
In 384 hole PCR reaction plates, add 2 μ L, sealer, centrifugation per hole.384 orifice plates for having added in iPlex reaction solutions are put into PCR instrument,
Run the response procedures of entitled " extension ".Extension condition is：94 DEG C of 30sec, 40 cycle 94 DEG C of 5sec, 5
Partial circulating (52 DEG C of 5sec, 80 DEG C of 5sec) }, 72 DEG C of 3min.
3) reaction terminates, and the of short duration centrifugation of 384 orifice plates taking-up is spare, can be in 4 DEG C of preservations.
5th, product purification
1) 6mg resins are taken on 384 hole resin scraper plates, uniform fold scrapes off extra resin, places 20min.
2) the 384 orifice plate 1000rpm centrifugation 1min terminated will be reacted, 25 μ L deionized waters is added in per hole, are upside down in resin
(pay attention to fixing, it is impossible to shift) above plate, then invert and resin plate is buckled on 384 orifice plates, percussion makes resin fall into 384 holes
Plate, sealer.
3) using the long axis of 384 orifice plates as axle center, 384 orifice plate of overturning 20 minutes is spare after 3500rpm, centrifugation in 5 minutes.
6th, Mass Spectrometer Method
1) NanodispenserSpectroCHIP chips point sample
Detection sample is transferred to the MassARRAYSpectroCHIP chips of surface covering matrix from 384 hole reaction plates.
2) MassARRAY Analyzer Compact Mass Spectrometer Methods
After transferring the sample into SpectroCHIP chips, you can be put into mass spectrograph and be detected, each test point only needs 3-
5 seconds, automatical analysis.
3) TYPER softwares analysis experimental result, obtains typing data.
The present invention is described in more detail below by specific embodiment, for a better understanding of the present invention,
But following embodiments are not intended to limit the scope of the invention.
Flight mass spectrometer used is MassARRAYCompact System (SEQUENOM companies) in following embodiments, type
Number PT009044.Point sample instrument is MassArray TM Nanodispenser (SAMSUNG companies).
Embodiment 1：
Using time-of-flight mass spectrometry technology detect 10 renal transplant recipients PPIA, PPP3CB, PPP3R1, NFATC1,
Totally 5 genes amount to the genotype of 78 SNP sites to NFATC2.
1st, people's Whole Blood Genomic DNA is extracted
Whole Blood Genomic DNA is extracted, and adjust a concentration of 10-100ng/ul with DNA extraction kit.
2nd, multi-PRC reaction
1) objective gene sequence is searched, for mutational site, designs and synthesizes PCR primer
While 78 SNP sites in PPIA, PPP3CB, PPP3R1, NFATC1, NFATC2 gene are measured, the primer of design,
It is shown in Table 1.
2) according to sample 384 hole reaction tables of establishment have been extracted, the number per the corresponding DNA sample in hole, the primer are indicated.
3) 1 μ L DNA profilings are separately added into each hole of 384 orifice plates by table, pad pasting is spare after centrifugation in 2000rpm/10 seconds.
4) according to the form below prepares PCR reaction solution：
* divide and carry out multiplex PCR for 4 reaction systems, specific grouping is shown in Table 1.
5) 12 union of a row is taken, the PCR reaction solution 133 μ L being configured are added in per hole, it is spare after of short duration centrifugation.
6) PCR reaction solution 4ul in 12 unions is taken with the 10ul volley of rifle fires, adds in 384 orifice plates for having 1uL DNA, make each hole
Final volume is 5ul.
7) PCR instrument will be put into after the of short duration centrifugation of 384 orifice plates equipped with 5ul reaction solutions, runs the reaction interval of entitled " PCR "
Sequence.PCR response procedures are：94 DEG C of 15min, 45 cycles (94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min), 72 DEG C of 3min.
8), will be spare after the of short duration centrifugation of 384 orifice plates after PCR response procedures, it can be in 4 DEG C of preservations.
3rd, SAP processing
1) SAP reaction solutions are prepared
2) 12 union of a row is taken, SAP reaction solutions with every 66 μ L of hole are dispensed, after of short duration centrifugation, are sub-packed in the 10ul volley of rifle fires
384 hole PCR reaction plates add 2 μ L, sealer, centrifugation per hole.
3) 384 orifice plates for having added in SAP reaction solutions are put into PCR instrument, run the response procedures of entitled " SAP ".SAP is anti-
Answer program：37 DEG C of 40min, 85 DEG C of 5min.
4) reaction terminates, and the of short duration centrifugation of 384 orifice plates taking-up is spare, can be in 4 DEG C of preservations.
4th, extension
1) iPlex reaction reagents are prepared
2) 12 union of a row is taken, iPlex reaction solutions are dispensed with every 66 μ L of hole, after of short duration centrifugation, is dispensed with the 10uL volley of rifle fires
In 384 hole PCR reaction plates, add 2 μ L, sealer, centrifugation per hole.384 orifice plates for having added in iPlex reaction solutions are put into PCR instrument,
Run the response procedures of entitled " extension ".Extension condition is：94 DEG C of 30sec, 40 cycle 94 DEG C of 5sec, 5
Partial circulating (52 DEG C of 5sec, 80 DEG C of 5sec) }, 72 DEG C of 3min.
3) reaction terminates, and the of short duration centrifugation of 384 orifice plates taking-up is spare, can be in 4 DEG C of preservations.Each site single base extension product
It is shown in Table 1.
5th, product purification
1) 6mg resins are taken on 384 hole resin scraper plates, uniform fold scrapes off extra resin, places 20min.
2) the 384 orifice plate 1000rpm centrifugation 1min terminated will be reacted, 25 μ L deionized waters is added in per hole, are upside down in resin
(pay attention to fixing, it is impossible to shift) above plate, then invert and resin plate is buckled on 384 orifice plates, percussion makes resin fall into 384 holes
Plate, sealer.
3) using the long axis of 384 orifice plates as axle center, 384 orifice plate of overturning 20 minutes is spare after 3500rpm, centrifugation in 5 minutes.
6th, Mass Spectrometer Method
1) NanodispenserSpectroCHIP chips point sample
Detection sample is transferred to the MassARRAYSpectroCHIP chips of surface covering matrix from 384 hole reaction plates.
2) MassARRAY Analyzer Compact Mass Spectrometer Methods
After transferring the sample into SpectroCHIP chips, you can be put into mass spectrograph and be detected, each test point only needs 3-
5 seconds, automatical analysis.
3) TYPER softwares analysis experimental result, obtains typing data.
The gene of 78 SNP sites in 10 renal transplant recipients PPIA, PPP3CB, PPP3R1, NFATC1, NFATC2 genes
Type testing result is shown in Table 2.
In 1 PPIA, PPP3CB, PPP3R1, NFATC1, NFATC2 gene of table used in the genotype detection of 78 SNP sites
Primer and the Single base extension object of generation
| WELL | SNP_ID | Forward primer | Reverse primer | Single base extension primer |
| W1 | rs12958819 | ACGTTGGATGGTCCTCTGAAGAATATGCAC | ACGTTGGATGTTAAACCTGCAGCAGGAACG | CAGCTTACTCCACCATC |
| W1 | rs6096426 | ACGTTGGATGACAGCTAGCGCTGATATTAG | ACGTTGGATGAAGCTGGGATTATAAGCGTG | GTGCCCAGCCAGAATAT |
| W1 | rs6021266 | ACGTTGGATGAAGGCTTGCTTTGTGCCTAC | ACGTTGGATGCAGTGGTGAACTAAACAGGC | cAATGTCAGCTCCATGAA |
| W1 | rs68734 | ACGTTGGATGACAGAAACGTCAAGGGCTAC | ACGTTGGATGGGAAGGTTCTAGATGAAACG | aGAGGACTTGCACACAGA |
| W1 | rs6021262 | ACGTTGGATGCTGTGCTGATCTCACCAACG | ACGTTGGATGTATGGGGAATGTCTCACTGG | TCACCAACGTTGTATCACC |
| W1 | rs761376 | ACGTTGGATGTCTGGTTACGACCATGCTTC | ACGTTGGATGGGGCAATCGTGTAGAGAATT | TAACTAAGGCAGAACGTGG |
| W1 | rs747063 | ACGTTGGATGCTGAGATCAAGAGTCTGAGC | ACGTTGGATGAGTAACTTTCCCGCCATGAC | taaAGGTTTACATCCTGGCA |
| W1 | rs17199672 | ACGTTGGATGCCAAGGGTCCATCTTCTTTC | ACGTTGGATGTACAGGTCAGGCCAGATTCC | aTTCTTTCTTTTGCACCCTTT |
| W1 | rs2002311 | ACGTTGGATGAAAGGTTAACTGCAAACCCC | ACGTTGGATGTTCTGAGTGTTCACACAAAC | GCGAGTTTGGGACATATTTAC |
| W1 | rs6021276 | ACGTTGGATGAGATAAGCAGACAGATGTCC | ACGTTGGATGGCAGGGCTGGAATTGAAAAC | gaatACAGATGTCCAGAGAGA |
| W1 | rs2055899 | ACGTTGGATGCTCTTGGGATTTTTGGTTTTC | ACGTTGGATGCACCAGAGCTTCCCTTGTAG | ccctaCAGGCTCCAGCTGAGCG |
| W1 | rs9518 | ACGTTGGATGCCTGCGTTGTGTGTTTTCAG | ACGTTGGATGAATTGTACAGAGTGCCCAGC | gatTGCCCAGCAAGGCCTACAC |
| W1 | rs11696516 | ACGTTGGATGAGAGATGGTGCGAGGTTTAC | ACGTTGGATGAATTCCCATATGCCAGCTCC | TGGAGAGAACAGAGAGACTAAG |
| W1 | rs1017860 | ACGTTGGATGTCTTTTTAACCGTGTGAGGG | ACGTTGGATGGGAAAGCTGTTTCGTTAGAG | TTTCGTTAGAGATAAAAACACCA |
| W1 | rs12625064 | ACGTTGGATGCCATTTTACAGGTGGAAAGC | ACGTTGGATGAGCTTGCAGCCCCTTCCAGT | gcagtTGGAAAGCCTGAGTCATC |
| W1 | rs3787193 | ACGTTGGATGATTTCACATCACTCAGTTGC | ACGTTGGATGTCTTGTCGTTTTCTCATGGG | ggaATAGAATGGGGATTGGCGAC |
| W1 | rs4811189 | ACGTTGGATGGCCCTTCGAACCAGAAAAAC | ACGTTGGATGCCAAGGGTGTGTTTCTGATA | tttaTGATAAGATACAGTGCAGTT |
| W1 | rs12644 | ACGTTGGATGGGTTACCTAGATGTTATGAG | ACGTTGGATGAAAGCCAGCAGCAAATCACC | gggttTGAGTACTCAAGTTGCTTA |
| W1 | rs2426295 | ACGTTGGATGATCCAGCTAAGGTGTGTGTC | ACGTTGGATGCGTAAGAGAGATCAAAGGTG | cctcTTTGCAAAATCATTTTTGAGA |
| W1 | rs6021232 | ACGTTGGATGGACTACTAACAGGACATGCC | ACGTTGGATGTCCTAGATCTTTCTGGGTCG | ACTTTAAAAAATAGTAAGGTAGAAG |
| W1 | rs8123890 | ACGTTGGATGTAATGTATTGCGTGCTTGCT | ACGTTGGATGCTCATAGCGTTGCAGTGAAG | ccattTATTGCGTGCTTGCTAAATAC |
| W1 | rs6013189 | ACGTTGGATGTCTGTGCTCAGTTTCTCACC | ACGTTGGATGTCTGATCCCTCATAAGAGCC | tcgtgTCTCACCAGTAAAATTGAGAA |
| W1 | rs2426300 | ACGTTGGATGACTTGGTGCAGTCATGAAGG | ACGTTGGATGGTGAGACAATGAGACTGACC | ctgcTGGAATTAGTGGCCTAAGAAGA |
| W1 | rs6096431 | ACGTTGGATGGACATGAAGATGGGAACAGC | ACGTTGGATGGTACCCAATTGGTAGATCCC | ggggaAGCAGATACTGAGGACTAATG |
| W2 | rs6123048 | ACGTTGGATGATCAGGCAGCTCTTCTACAC | ACGTTGGATGGTAGACAGACGAGTGAATCC | CTTCAATGTGGGACTCTG |
| W2 | rs3787190 | ACGTTGGATGATTTGACCTTCTGGGGCTTC | ACGTTGGATGCACACGTTCTCATTGACTCC | TCTCTCCAATTTGCTTCTT |
| W2 | rs6067766 | ACGTTGGATGATGAAACTACAAAGGAGGGC | ACGTTGGATGGAGTGAGGTGCTCCTTTAAG | TATGGGTAGGTCAGCATCA |
| W2 | rs11086343 | ACGTTGGATGAGTGCCAGGCTGTGGAAACT | ACGTTGGATGCCAAAGAGTTGAGGGTTTCC | aGAAACTGCTGGAATCGGT |
| W2 | rs228835 | ACGTTGGATGGGCCAGGGTTCCCACTGAA | ACGTTGGATGAAATGTGTAACCCTTCAGGC | cCCCTTCAGGCAAAGTTCAC |
| W2 | rs754505 | ACGTTGGATGCCTGCTTGTTAAACTAAGTC | ACGTTGGATGCGAGGGCGGCACTTGGTAT | aaTTTATAGATGCGACTCCC |
| W2 | rs17199748 | ACGTTGGATGACACCCTGAAGATCTTGCTG | ACGTTGGATGGTCGATCTAAGACTTTGAGG | TGGAGGTACATGTGGTTAAG |
| W2 | rs761374 | ACGTTGGATGTTGAAGGGAGGAACTGTGAC | ACGTTGGATGTCCATCACTGACTCTGACCT | cgttCCTTCATGTCCCCATCT |
| W2 | rs4799055 | ACGTTGGATGATCCAGGACAGGAGTCTTTG | ACGTTGGATGACAGCGGTTTCTCAAATCAC | aTTCTCAAATCACCTAAAGGG |
| W2 | rs910156 | ACGTTGGATGAGGCTCTGAATCAAGCTTCC | ACGTTGGATGTGTTCCCTAGTACAGCACCC | gctcACTACTCAGCAACATCTT |
| W2 | rs11086340 | ACGTTGGATGGATGGATGGTAACATGTGGG | ACGTTGGATGGTGCGAAGGTTGAGAAATCC | TTAAGACATTGGAGTAAGAAAC |
| W2 | rs3894049 | ACGTTGGATGGGAACAAGACGGCTCTCGG | ACGTTGGATGATGGTGACAGTGACAGGGTG | ttaaGGCGTCTGAGGTCTCCATC |
| W2 | rs2024581 | ACGTTGGATGCGCCCCTCATTTTATGCAAG | ACGTTGGATGGAGCGCGGTTACTTAAAATG | GAAGCTGAGAATTACCTGTGTAT |
| W2 | rs306871 | ACGTTGGATGTGCATGCTGAGGCCTGAAC | ACGTTGGATGAGGAGCTAGTTTCCCAGTCG | ggcgGAGGCCTGAACGGCGGAGG |
| W2 | rs6021231 | ACGTTGGATGGTGTGGTGGCTCTAATCCAA | ACGTTGGATGTCCAGATGCTGCAGAAATCC | ttttcGAAATCCCATAGTCTCTCT |
| W2 | rs10249442 | ACGTTGGATGTACCTTTCTCGTCTTGGTTC | ACGTTGGATGATGCTTGCCATCCAACCTTG | tACCTTGAAAAGGAAGAACAATCA |
| W2 | rs6067796 | ACGTTGGATGGAAGAAAATCCAAACTGCCC | ACGTTGGATGTGTGGCTGGACTGGAGTTG | taaagCCAAACTGCCCATTGCAGCC |
| W2 | rs8090864 | ACGTTGGATGTGGAGTTACCTGGCAGATTG | ACGTTGGATGCATGGCTAGCTGAGATCTTC | tATTGGAATATTTCAGTTCTGTGTC |
| W2 | rs4396773 | ACGTTGGATGCCAAAGACTGACTAGCCATC | ACGTTGGATGGCACATGATCCTTCAACTGG | tttggTGTGCTGAAATGGAAACTAT |
| W2 | rs228832 | ACGTTGGATGCACAATCAGAGCAACACAGC | ACGTTGGATGTTGGGAGGTGGATTATACGG | cccatATTATACGGAACTTCTGGTCA |
| W2 | rs754093 | ACGTTGGATGAAGACCATACTACAGCCAGC | ACGTTGGATGTGGCATCAGGGTGCTTCTCT | gaaCATCAGGGTGCTTCTCTGCGGAC |
| W2 | rs2060611 | ACGTTGGATGCAACAACCGCACACTGCTG | ACGTTGGATGATGTTGGTTCACAGACGTGC | gttgtTTCACAGACGTGCGGTGGCGG |
| W2 | rs6021183 | ACGTTGGATGAGATCACAGAACCCCGTATC | ACGTTGGATGGAGGTCTGTGCCTTAATGAG | gtgcGAGGTTGGTGTTAAAATGTCAC |
| W3 | rs3826572-B-1 | ACGTTGGATGTGCTGCAGGTTTAATTACGC | ACGTTGGATGATCCGCCTCACTTTCCGTC | TCCCTTCTAAGGCCCAC |
| W3 | rs2581731 | ACGTTGGATGTCTTTGAGACGCACAGGATG | ACGTTGGATGTTCACGCTGGAGCCTCCTCT | GGGCTGTGCGTGACCTT |
| W3 | rs12994570 | ACGTTGGATGGAGAAGCTAAGTGGTTGTGG | ACGTTGGATGGCCTCTCAAATTACAACTAGG | TCTGTTCAGAAGCAATGA |
| W3 | rs12608349 | ACGTTGGATGTAGTTCCACATACGCCGTTG | ACGTTGGATGGAAAACAGACACAGCCCGAC | CCGTTGTTCCCGCAGCCCA |
| W3 | rs9638978 | ACGTTGGATGGTGCCCACCCAATTAAGTGC | ACGTTGGATGAAAACAAAACAAAAACCCC | gACAAAAAACCCAACCAAC |
| W3 | rs7560138 | ACGTTGGATGCCCAGCAAACTGGAAGTTTC | ACGTTGGATGTTTGTTGAGCACCTACTTGG | agGGAAGTTTCTCGAGGAC |
| W3 | rs3787189 | ACGTTGGATGTTATTCCTGTTCGACGAGGG | ACGTTGGATGTTCATTGCTGACCAGCTGCC | CAGCTGCCTGACTTTAGGTA |
| W3 | rs6021240 | ACGTTGGATGTCACGTTCTCATCAACCTCC | ACGTTGGATGAAGGGACAAGGCAGAAATTA | AGGGGTCCAGGTGAGAGATG |
| W3 | rs177820 | ACGTTGGATGAAAGGGGCATGATAGATGTG | ACGTTGGATGCTAAATGTGTATGTCAGATGG | cCTGACTGTTTTATGGTTTCA |
| W3 | rs6463247 | ACGTTGGATGCATCCCTAAGATACTAGAAG | ACGTTGGATGCTAAGGGCCAAGTTTCTATC | gTCACATTGGCTATATTTGTC |
| W3 | rs2596606 | ACGTTGGATGCTGCATTTTGATGATGATTC | ACGTTGGATGTATCACCCGTCAAAAGCTGG | GATGATGATTCAATGGTGTTA |
| W3 | rs6126240 | ACGTTGGATGGGAGGAAGCTCACCAAGATG | ACGTTGGATGCAGCTACCACCTCTATACTC | ggaccAGCACTGGAATTCCAAG |
| W3 | rs7240932 | ACGTTGGATGAGCCATTTTAGAGCTCAGCG | ACGTTGGATGTAGCAGGTGGCCTGTGATTC | acgtgAGAGCTCAGCGGAATGA |
| W3 | rs2036892 | ACGTTGGATGGGCTCATATCCTACGAATCC | ACGTTGGATGCTTAGGACTAGCTGTGTACC | ACCAGAATCTTCTGTTTTGTTCT |
| W3 | rs3826567 | ACGTTGGATGACAAAACCACACGCCACGG | ACGTTGGATGTAGGTTCCAAGGCAGACTCA | GGCAGACTCATAAAAAATTAATCC |
| W3 | rs228840 | ACGTTGGATGTTTCAACCATGGGCAGCTCG | ACGTTGGATGACCTCCTCTTTTGAACAGAC | gaaatAGAGAGAAGTCACTGTGAT |
| W3 | rs6123045 | ACGTTGGATGTGCTGCATTTTGACCATGGC | ACGTTGGATGCCCAAACAGTAAAGATAAAAC | ttGCACCATGCCCTTGCTAAACTAT |
| W3 | rs2280055 | ACGTTGGATGAGGTCCCCTCGTTGGCACAG | ACGTTGGATGCTAGTGCCATCGACAGACAG | tcaaTCAGCCACAGAAAGACCCCAC |
| W3 | rs3735481 | ACGTTGGATGGATCAGTTGTAATTATAGC | ACGTTGGATGGGAACGGAATCGTACTCAAT | CGGAATCGTACTCAATATTTAACTTT |
| W3 | rs3787197 | ACGTTGGATGATGATAGTGAGCTTAGGCAG | ACGTTGGATGGAGTGATTCATTCTCCAGCG | gggatTGAGCTTAGGCAGACTCCAAA |
| W4 | rs6506775 | ACGTTGGATGTGCACTCAGCAGCACAGTGG | ACGTTGGATGCACAACGCAGAGAGAAAGAC | CGTGCGACCCCCACA |
| W4 | rs228852 | ACGTTGGATGTCCTGAGCTCAAGCGATCGT | ACGTTGGATGAAAAATACTGATGCCCAGCC | AGCGATCGTCCGGCCT |
| W4 | rs2426312 | ACGTTGGATGTTCCCATCAATGCGCATCTC | ACGTTGGATGGGCTTTATCAGAACAGCTTG | AGGGTAGAGAAGTGCC |
| W4 | rs8091347 | ACGTTGGATGTCTTTCTCGCTGGCAGATTG | ACGTTGGATGAAACGCTGCCTCACAGTGG | GCTGTCTGTCATGCCGC |
| W4 | rs12959273 | ACGTTGGATGTGTGTCCTTGCCAAACTGTC | ACGTTGGATGCCAATCAACAACCAATTGTGC | CTGTCTGGTTTGTTTAGC |
| W4 | rs12569942 | ACGTTGGATGATGAGGCCTCTGACTTCAAC | ACGTTGGATGGAGATTAACTGACCTTAGGC | CAGTCTGTGCCAAGTGAAT |
| W4 | rs228851 | ACGTTGGATGATAGGACAGAGACTGACAAG | ACGTTGGATGTGTTTAGATGGCTCCTCAAG | cgcaCCCCTTTGTGCTCTGA |
| W4 | rs4811187 | ACGTTGGATGTCAAGAGACTTCCCTGATGC | ACGTTGGATGTCACAAGTACCAACCTGTGC | ctaCCCATCCTCTCAACTTTA |
| W4 | rs160189 | ACGTTGGATGACACACTTGGAGACACCCTG | ACGTTGGATGAGGATCAGATGAGGGAAGGC | GCTCGGCTCTCACTGCAGAGT |
| W4 | rs2426302 | ACGTTGGATGAGTGAGCCTCGTGAGAAGTG | ACGTTGGATGCCTGGCCAGTATTGAATTTT | GTATGAAAGATAACTTTTAGCAG |
| W4 | rs6021264 | ACGTTGGATGCCACCTTGATAAAGACAGTG | ACGTTGGATGAAAGAAGGGACAGCAGTTAG | TAGAACTAACATAAGCTTCATTAA |
* it notes：W1, W2, W3, W4 represent 4 multi-PRC reaction systems respectively.
78 SNP sites in 2 10 renal transplant recipients PPIA, PPP3CB, PPP3R1, NFATC1, NFATC2 genes of table
Genotype call results
Specific embodiments of the present invention are described in detail above, but it is intended only as example, it is of the invention and unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
It substitutes also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (4)
1. measuring the method for people's cyclosporine action target spot gene pleiomorphism while a kind of non-diagnostic and therapeutic purposes, feature exists
In including the following steps：
Step 1：The genomic DNA of peripheral blood cells sample is extracted, is drawn for the different mutational site design screening of target gene
Object；And it is combined into different PCR reaction systems and carries out multi-PRC reaction；For PPIA, PPP3CB, PPP3R1, NFATC1,
The primer of 78 SNP site designs is as shown in the table in NFATC2 genes：
Step 2：After the PCR product SAP processing of amplification, single base extension is carried out；
Step 3：After carrying out chip point sample, it is detected with Matrix-assisted laser desorption ionization；
Step 4：Testing result is analyzed using software, obtains typing data；
Wherein, genomic DNA is extracted using DNA extraction kit in the step 1, and the DNA concentration of extraction is adjusted to 10-
100ng/ul；The PCR response procedures are：94℃15min；45 cycles：94 DEG C of 20sec, 56 DEG C of 30sec, 72 DEG C of 1min；72
℃3min。
2. method that is according to claim 1 a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, feature
It is that the SAP response procedures are：37 DEG C of 40min, 85 DEG C of 5min.
3. method that is according to claim 1 a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, feature
It is that the single base extension condition is：94℃30sec；40 cycles：94 DEG C of 5sec, 5 partial circulatings：52 DEG C of 5sec,
80℃5sec；72℃3min.
4. method that is according to claim 1 a kind of while measuring people's cyclosporine action target spot gene pleiomorphism, feature
It is between the step 2 and step 3 that also there is the step of product purification, the operation of product purification is：
(1) resin uniform fold on resin scraper plate is taken, places 20min；
(2) the orifice plate 1000rpm that end is reacted in step 2 is centrifuged into 1min, 25 μ L deionized waters is added in per hole, are upside down in resin
It above plate, then inverts and resin plate is buckled on orifice plate, percussion makes resin fall into orifice plate, sealer；
(3) overturning orifice plate 20 minutes, 3500rpm, centrifugation 5min.
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