CN108676857A - Detect the primer and method of CYP2C8 point mutation - Google Patents

Detect the primer and method of CYP2C8 point mutation Download PDF

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Publication number
CN108676857A
CN108676857A CN201810576148.8A CN201810576148A CN108676857A CN 108676857 A CN108676857 A CN 108676857A CN 201810576148 A CN201810576148 A CN 201810576148A CN 108676857 A CN108676857 A CN 108676857A
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cyp2c8
primer
sequence
sequencing
sites
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桑志高
吴鹏飞
王淑
王淑一
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HANGZHOU ADICON CLINICAL LABORATORY CENTER Co Ltd
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HANGZHOU ADICON CLINICAL LABORATORY CENTER Co Ltd
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract

The invention discloses a kind of specific primers and method of CYP2C8*2 and CYP2C8*3 partings comprising 1 pair of primer of amplification CYP2C8*2 polymorphic sites；Expand 2 pairs of primers of CYP2C8*3 polymorphic sites.Using PCR amplification and Sanger sequencing technologies, the present invention can rapidly come out the relevant site primer of CYP2C8 partings.The testing result completed using the present invention is accurate, and clinical individual administration and the structure optimization of new drug candidate compound can be carried out for clinical pharmacology scholar and is further screened to provide useful information.

Description

Detect the primer and method of CYP2C8 point mutation
Technical field
It is a kind of prominent using PCR and Sanger PCR sequencing PCRs detection CYP2C8 points the invention belongs to medicine and biotechnology The method and primer of change.
Technical background
Cytochromes super families are a major class hemoproteins, universally present in various life entities, are aoxidizing generation It plays a significant role in terms of thanking to numerous life entity xenobiotics.Different individuals are for drug and other toxic substances Matter will appear different reactions, and many this othernesses have been found to be since the gene pleiomorphism of human body cell cytochrome p 450 draws It rises.In addition, the gene pleiomorphism shape for being responsible for the Cytochrome P450 of metabolism life entity endogenous material is also certain specific something lost Pass the pathogenesis of disease.CYP2C8 accounts for about 7% in human liver's microsomal cytochrome, is at least responsible for facing for metabolism 5% Bed drug.A variety of important drugs all can be used as the substrate of CYP2C8, including anticancer drug, arrhythmia drug, hypoglycemic Drug, antiepileptic, HMG-CoA reductase inhibit drug etc..The research of CYP2C8 gene pleiomorphisms is for realizing medication Bodyization can play an important role.In addition, some specified diseases caused by CYP2C8 gene pleiomorphisms have also appeared in the newspapers.To mesh Before until, have more than 10 kinds of CYP2C8 gene pleiomorphism be reported, the wherein more a height of mutant CYP2C8*2 of occurrence frequency (I269F) and mutant CYP2C8*3 (R139K and K399R).The mutation of CYP2C8*2 is happened on 5 exons, the 269th Amino acid is phenylalanine by isoleucine mutation.Its occurrence frequency has apparent ethnic difference anisotropic, prominent in African Frequency is 15%-20%, rarely has or do not find the mutant in Europe, Asian.The mutation of CYP2C8*3 is two SNP Close linkage, be respectively occurring on No. 3 and 8 exons, the 139th amino acids sport lysine by arginine, the 399th Position lysine mutation is arginine.CYP2C8*3 is one of the gene pleiomorphism of most common CYP2C8, is primarily present in white man In (gene frequency 10%-23%), Africa and asian population in it is more rare.Carry CYP2C8*3 homozygous mutations (A/A) scavenging capacity of taxol is substantially reduced with heterozygous mutant (G/A) genotype patient, about wild type patient 15%, this may cause drug more to accumulate in vivo, therefore patients of the CYP2C8*3 with A should suitably reduce dosage. The inter-individual difference of the 6 ɑ hydroxylation activities of taxol of CYP2C8 leads to the clinical therapeutic efficacy and drug of taxol up to 38 times Safety is irregular.Therefore, there is highly important academic significance and clinical meaning for the research of CYP2C8 Genotypings.
The characteristics of in view of CYP2C8 genes in patients, Genotyping situation may instruct to provide important for patient medication Analysis foundation, be the promising technical research direction of tool by analyzing the mutation of CYP2C8 gene-correlations to carry out prognosis evaluation.Cause This, the technical issues of how quickly comprehensively obtaining the data of detection subject gene parting, become current urgent need to resolve.
Invention content
The object of the present invention is to provide the primer of CYP2C8 Genotypings and method, integrated application PCR amplification and gene are surveyed Sequence technology can be used for quickly detecting detection two kinds of Common genes types of CYP2C8*2 and CYP2C8*3.The detection CYP2C8 genes The primer of polymorphism, including：
The primer of CYP2C8 genes is expanded, base sequence is：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Further, further include sequencing primer, base sequence is：
Detection CYP2C8 genes sequencing primer base sequence be：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Further, primer sequence CYP2C8-1F/CYP2C8-1R is the primer for expanding the sites CYP2C8*2 I269, is drawn Object sequence C YP2C8-2F/CYP2C8-2R and CYP2C8-3F/CYP2C8-3R are the amplification sites CYP2C8*3 R139 and K399 The primer of point.
The present invention also provides the methods of detection CYP2C8 gene pleiomorphisms, include the following steps：
1. extracting the genomic DNA in peripheral blood；
2. with the DNA extracted in PCR amplification step 1；
3. the amplified production in pair step 2 is sequenced；
4. a pair sequencing result judges, determine whether CYP2C8 genes mutate；
Wherein PCR amplification primer is：
The primer of CYP2C8 genes is expanded, base sequence is：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Further, further include sequencing primer, base sequence is：
Detection CYP2C8 genes sequencing primer base sequence be：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Advantageous effect：The specificity that the present invention devises amplification CYP2C8*2 and CYP2C8*3 parting polymorphic sites is drawn Object.Using round pcr, stable amplification system is constructed.By adjusting reaction conditions such as primer concentration, annealing temperatures, can make Amplification efficiency reaches best.Primer of the present invention can all amplify CYP2C8*2 the and CYP2C8*3 polymorphic sites Come, the case where no matter the where position of these polymorphic sites mutates, is all not in missing inspection ensured.Detection process is quick Sensitive, easy to operate and accuracy is high, and testing result is clearly clear；High specificity is detected, it is prominent using most accurate gene at present Become detection method, purpose is strong；Detection sample blood sampling volume is few, and the experiment equipment used is less, cost-effective.
Description of the drawings
Fig. 1 is the I269 for carrying out screening CYP2C8*2 to first case blood sample to be checked using primer sequencing approach of the present invention Site sequencer map.(frame selected bit is set to the sites I269).
Fig. 2 is the R139 for carrying out screening CYP2C8*3 to second case blood sample to be checked using primer sequencing approach of the present invention Site sequencer map.(frame selected bit is set to the sites R139).
Fig. 3 is the K399 for carrying out screening CYP2C8*3 to third example blood sample to be checked using primer sequencing approach of the present invention Site sequencer map.(frame selected bit is set to the sites K399).
Specific implementation mode
Embodiment 1
Extract the DNA in blood：1) 300 μ l blood are extracted and 900 μ l erythrocyte cracked liquids is added, overturn mixing, room temperature is put It sets 5 minutes, during which overturns mixing several times again.12,000rpm centrifugation 1min, suck supernatant, leave leukocyte cell pellet, add 200 μ l Buffer solution GA, oscillation to thorough mixing.2) 20 μ l Proteinase K Solutions, mixing is added.3) 200 μ l buffer solution GB are added, fully run Mixing, 70 DEG C are placed 10 minutes, and solution strain is limpid, and brief centrifugation is to remove the droplet of cap wall.4) be added 200 μ l without Water-ethanol, fully vibrates mixing 15 seconds, at this time it is possible that flocculent deposit, brief centrifugation is to remove the droplet of cap wall. 5) previous step acquired solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column is put into collecting pipe), 12, 000rpm is centrifuged 30 seconds, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.6) 500 μ l bufferings are added into adsorption column CB3 Liquid GD (please first checks whether before use and absolute ethyl alcohol has been added), and 12,000rpm centrifugations 30 seconds outwell waste liquid, by adsorption column CB3 It is put into collecting pipe.7) 700 μ l rinsing liquids PW are added into adsorption column CB3 (please first to check whether before use and anhydrous second has been added Alcohol), 12,000rpm centrifugations 30 seconds outwell waste liquid, adsorption column CB3 are put into collecting pipe.8) 500 are added into adsorption column CB3 Waste liquid is outwelled in μ l rinsing liquids PW, 12,000rpm centrifugations 30 seconds.9) adsorption column CB3 is put back in collecting pipe, 12,000rpm centrifugations 2 minutes, outwell waste liquid.Adsorption column CB3 is placed in and is placed at room temperature for several minutes, thoroughly to dry rinsing remaining in sorbing material Liquid.10) adsorption column CB3 is transferred in a clean centrifuge tube, it is slow that 100 μ l elutions is vacantly added dropwise to the intermediate position of adsorbed film Fliud flushing TE is placed at room temperature for 2-5 minutes, and solution is collected into centrifuge tube by 12,000rpm centrifugations 2 minutes.
Wherein, 10 × erythrocyte splitting formula of liquid is：NH4Cl 82g, NaHCO38.4g, EDTA-Na23.72g Add ddH2O is settled to 1000ml.
Embodiment 2
PCR amplification：
PCR amplification system preparation of reagents method is as follows：
Reaction condition is as follows：
Wherein, primer sequence is：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Embodiment 3
PCR product purifies：Enzyme purification presses following system configurations：CIP (NEB companies) 0.1 μ l, Exo I (NEB companies) 0.5 μ L, 1.4 μ l of deionized water；It is eventually adding 9 μ l PCR products.It is as follows that it purifies response procedures：37 DEG C of 50min, 95 DEG C of 5min.
Embodiment 4
Sanger sequencing reactions：Per 5 μ l of reaction system：Bigdye adds 1 μ l, 3.2P primers that 1 μ l, template after purification is added to add 2 μ l are eventually adding 1 μ l ddH2O.The of short duration centrifugation of lid upper tube cap low speed, vortex mixing, the again of short duration centrifugation of low speed.Upper PCR instrument Sequencing reaction is carried out, following condition is pressed in reaction：95 DEG C of pre-degeneration 4min；95 DEG C of denaturation 15s, 50 DEG C of annealing 20s, 60 DEG C extend 2min, 25 cycles.The Sanger sequencing primers include：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA -3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC -3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA -3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG -3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT -3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA -3’
Embodiment 5
Product purification is sequenced：The EDTA of 2 μ l 125mmol is added into the product for completing sequencing reaction, stands 5min；Add Enter 15 μ l absolute ethyl alcohols, whirlpool mixing；3700rpm centrifuges 30min；It is inverted centrifugation 15sec, 50 μ l, 70% ethyl alcohol, whirlpool is added Mixing；3700rpm centrifuges 15min；It is inverted centrifugation 15sec, is stored at room temperature 30min, fully volatilize ethyl alcohol；10 μ l are added per hole Hi-Di, sealing plate are placed on 95 DEG C of pre-degeneration 5min in PCR instrument, are put into -20 DEG C of refrigerators cooling 5min rapidly；Upper sequenator is surveyed Sequence.
The sequencing result of measuring samples is compared with wild type standard sequence, is open country if consistent with standard sequence Raw type, inconsistent with standard sequence is then mutation.
Embodiment 6
To first case whole blood sample to be checked by the extraction for carrying out DNA described in embodiment 1-5, PCR amplification is sequenced, obtains Peak figure such as Fig. 1 is sequenced, obtaining the sites I269 sequencing result sequence is：ATC.According to result criterion, the sites sample I269 It is unmutated.
Embodiment 7
To second case whole blood sample to be checked by the extraction for carrying out DNA described in embodiment 1-5, PCR amplification is sequenced, obtains Peak figure such as Fig. 1 is sequenced, obtaining the sites R139 sequencing result sequence is：AGG.According to result criterion, the sites sample R139 It is unmutated.
Embodiment 8
To third example whole blood sample to be checked by the extraction for carrying out DNA described in embodiment 1-5, PCR amplification is sequenced, obtains Peak figure such as Fig. 1 is sequenced, obtaining the sites K399 sequencing result sequence is：AAA.According to result criterion, the sites sample K399 It is unmutated.
From testing result as can be seen that primer of the present invention can amplify the polymorphism of CYP2C8*2 and CYP2C8*3 Site areas sequence, and sequencing result entirely accurate quickly can carry out Genotyping to CYP2C8*2 and CYP2C8*3.
Sequence table
<110>ADICON Clinical Laboratories, Inc.
<120>Detect the primer and method of CYP2C8 point mutation
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ttcaatcagg gcttggtgta 20
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tatttcaaga agagggtcta tgc 23
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cactttcttc cttggctgtg a 21
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gaccttttat tcctacaccc tatg 24
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tgtctggctg gacctgagtt 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
ggtggagcac atgggtgtta 20

Claims (4)

1. a kind of primer being detected CYP2C8 gene polymorphism sites using Sanger PCR sequencing PCRs using DNA as template, feature are existed In including：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA-3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC-3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA-3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG-3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT-3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA-3’
The nucleotide sequence of the sequencing primer includes：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA-3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC-3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA-3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG-3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT-3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA-3’。
2. the primer of detection CYP2C8 gene polymorphism sites according to claim 1, which is characterized in that primer sequence CYP2C8-1F/CYP2C8-1R is the primer for expanding CYP2C8*2 polymorphic sites, primer sequence CYP2C8-2F/CYP2C8-2R It is the primer for expanding CYP2C8*3 polymorphic sites with CYP2C8-3F/CYP2C8-3R.
3. the primer of detection CYP2C8 gene polymorphism sites according to claim 1, which is characterized in that primer sequence CYP2C8-1F/CYP2C8-1R is the primer that CYP2C8*2 gene amplification products are sequenced, primer sequence CYP2C8-2F/CYP2C8- 2R and CYP2C8-3F/CYP2C8-3R is the primer that CYP2C8*3 gene amplification products are sequenced.
4. a kind of method of detection CYP2C8 gene polymorphism sites, which is characterized in that include the following steps：
(1) genomic DNA in peripheral blood is extracted；
(2) with the DNA extracted in PCR amplification step 1；
(3) amplified production in step 2 is sequenced；
(4) sequencing result is judged, determines whether CYP2C8 genes mutate；
Wherein PCR amplification primer is：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA-3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC-3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA-3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG-3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT-3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA-3’
Sequencing primer base sequence is：
CYP2C8-1F 5’-TTCAATCAGGGCTTGGTGTA-3’
CYP2C8-1R 5’-TATTTCAAGAAGAGGGTCTATGC-3’
CYP2C8-2F 5’-CACTTTCTTCCTTGGCTGTGA-3’
CYP2C8-2R 5’-GACCTTTTATTCCTACACCCTATG-3’
CYP2C8-3F 5’-TGTCTGGCTGGACCTGAGTT-3’
CYP2C8-3R 5’-GGTGGAGCACATGGGTGTTA-3’。
CN201810576148.8A 2018-06-06 2018-06-06 Detect the primer and method of CYP2C8 point mutation Pending CN108676857A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102912005A (en) * 2011-08-02 2013-02-06 广州益善生物技术有限公司 CYP2C8 gene polymorphism detection specific primers and liquid chip
CN104673898A (en) * 2015-02-02 2015-06-03 武汉艾迪康医学检验所有限公司 Primer capable of detecting CYP2C9*2 gene mutation, and detection method
CN106222281A (en) * 2016-08-10 2016-12-14 中南大学湘雅三医院 Test kit, application and method of based on the gene pleiomorphism accurate medication of guiding children patient
CN107841557A (en) * 2016-09-20 2018-03-27 宁波美丽人生医药生物科技发展有限公司 Drug taxol curative effect related gene genetic polymorphism detection kit and detection method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102912005A (en) * 2011-08-02 2013-02-06 广州益善生物技术有限公司 CYP2C8 gene polymorphism detection specific primers and liquid chip
CN104673898A (en) * 2015-02-02 2015-06-03 武汉艾迪康医学检验所有限公司 Primer capable of detecting CYP2C9*2 gene mutation, and detection method
CN106222281A (en) * 2016-08-10 2016-12-14 中南大学湘雅三医院 Test kit, application and method of based on the gene pleiomorphism accurate medication of guiding children patient
CN107841557A (en) * 2016-09-20 2018-03-27 宁波美丽人生医药生物科技发展有限公司 Drug taxol curative effect related gene genetic polymorphism detection kit and detection method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
柳海平等: "痰液质酒精性股骨头坏死CYP2C8基因rs17110453位点多态性研究", 《中国中医药信息杂志》 *
汪峻等主编: "《基因操作技术》", 31 August 2010, 华中师范大学出版社 *

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Application publication date: 20181019