CN106554995A - The method and test kit of detection chemotherapy of tumors personalized medicine related gene - Google Patents
The method and test kit of detection chemotherapy of tumors personalized medicine related gene Download PDFInfo
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Abstract
The invention provides the method and test kit of a kind of detection chemotherapy of tumors personalized medicine related gene.Compared with prior art, the present invention has advantages below：1) the reagent consumptive material for using is relatively easy and stable, it is not necessary to the expensive reagent such as fluorescent dye, special enzyme；2) reaction can be carried out in micro system, reduce the use of sample and various consumable goodss；3) due to the molecular weight (mass-to-charge ratio) and the direct type (needing not move through any type of signal conversion) for determining base of mass-spectrometric technique direct detection DNA, therefore, as long as having an amplification of DNA fragments for copying to be amplified in theory to can recognize that, so as to high sensitivity；4) the features such as mass-spectrometric technique also has automatization, high throughput testing, therefore, mass-spectrometric technique is combined with many primer extension techniques, can detect multiple gene locis simultaneously, so as to greatly reduce workload, improve detection flux in a reaction system.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to multiplexed PCR amplification technology and ground substance assistant laser solution
Analysis ionization time-of-flight mass-spectrometric technique (MALDI-TOF-MS).And more particularly it relates to detect that chemotherapy of tumors is individual
Change the method and test kit of medication related gene.
Background technology
Chemotherapeutics are the medicines to malignant tumor associated diseases.It is numerous that these medicines can act on growth of tumour cell
In the different links grown, tumoricidal cell cycle suppresses or kills tumor cell.Such as 5-fluorouracil, cisplatin
Tumor cell is killed by disturbing the mechanism such as DNA of tumor cell synthesis, destruction DNA of tumor cell Deng front-line chemotherapeutic agents.Chemotherapy
Drug therapy is to treat one of Main Means of tumor at present.
The antineoplastic chemotherapy medicine of Clinical practice has different degrees of toxic and side effects, and they are in the same of killing tumor cell
When, the cell of normal structure is killed again.Toxicity is the immediate cause for limiting chemotherapeutics dosage or using.The curative effect of chemotherapy
It is anti-with individual patient difference (patient is to chemotherapy medicament sensitive degree and the tolerance degree to medicine) and the toxicity of medicine itself
Should be relevant.In recent years, a large amount of clinical researches show that each chemotherapeutics has the corresponding target for assessing its effect, change
The curative effect for treating medicine is mainly related to the polymorphism of related gene and expression, and such as CDA gene pleiomorphisms affect gemcitabine
Curative effect, UGT1A1 is related to the toxic and side effects of irinotecan etc..The expression and polymorphism of these genes can be can help
The toxic and side effects risk for helping doctor to be all kinds of chemotherapeutics of patient evaluation, is that patient formulates that toxic and side effects are minimum, curative effect is optimal
Chemotherapy regimen.
Specific personalized medicine related gene is as follows：
1st, the personalized medicine related gene of gemcitabine
Gemcitabine is a kind of Difluoronucleosides class antimetabolic antineoplastic agent of destruction cellular replication, is clinically widely used in
The first-line treatment of nonsmall-cell lung cancer, simultaneously for including its including cancer of pancreas, carcinoma of gallbladder, breast carcinoma and leiomyosarcoma etc.
His many solid tumors are active.But the therapeutic window of the medicine is little, easily cause the untoward reaction based on bone marrow depression.
CDA gene pleiomorphisms can affect the pharmacokineticss of gemcitabine, by damaging removing toxic substances work(of the CDA to gemcitabine
Can, cause poisonous side effect of medicine to increase, the cancer patient of CDA reduced activities is receiving to be easily caused more high poison when gemcitabine is treated
Property occur.There is two kinds of gene pleiomorphisms, 79A in CDA>C and 208G>A, its saltant type can cause CDA reduced activities, suffer from tumor
Person is receiving easy generation higher toxic and side effects when gemcitabine is treated.
2nd, the personalized medicine related gene of platinum medicine (cisplatin, carboplatin, oxaliplatin)
Platinum medicine (cisplatin, carboplatin, oxaliplatin) is clinically the most frequently used cycle non-specific antitumor drug, is made
Major target class is DNA.Platinum medicine enters cell, and combining to form Pt-DNA adducts with DNA in tumor cell causes DNA
Structural change DNA replication dna, transcription obstacle, cause death of neoplastic cells.Platinum medicine resistance mechanism mainly includes：DNA repairs energy
Power strengthens, drug detoxication increases, reduce ingestion of medicines accumulation, body is improved to the toleration of platinum class-DNA complex etc., is related to
Several genes, albumen and signal transduction pathway.
(c.580C research show XRCC1>T, c.1196A>G) and GSTP1 (c.313A>G) gene mutation and medication effect
It is closely related.
XRCC1 participates in the BER (base excision repair approach) caused because of ionizing radiation and oxidative damage and singly-bound ruptures and repaiies
Multiple, the stability to maintaining gene plays pivotal role while relevant with platinum medicine opposing.Glutathione transferase P1
(GSTP1) be cell antibody Monoclonal anticancer become main detoxification system.The presence of GSTP1 gene pleiomorphisms can cause the phase which is expressed
Answering the active difference of enzyme, to cause function of detoxification to change closely related with the curative effect of platinum-based chemotherapy.Outside on GSTP1 genes the 5th
Show genetic polymorphism A in 105 sites of son>The G transformer effects activity of GSTP1 albumen, causes biologies of the GST to platinum medicine
Conversion capability is reduced, so as to improve clinical Benefit rate.
3rd, the personalized medicine related gene of fluorouracil medication
Fluorouracil drug is worked with antimetabolite, is converted into effective fluorouracil deoxidation core in the cell
After thuja acid, thymidylic acid is converted into by intracellular thymidylate synthetase by blocking deoxyribouridine acid, and disturbs the conjunction of DNA
Into.Fluorouracil equally can be with the synthesis of RNA interfering.
Dihydropyrimidine dehydrogenase (DPD) is the key enzyme in 5-Fu metabolic processes, and DPD enzymatic activitys have individuality in crowd
Difference, and affected by DPD enzyme genes (DPYD) polymorphism.DPYD gene 1905+1G>A mutation are almost wholly constrained to DPD enzymes
The low patient of activity, the enzymatic activity lacks to cause to remove in 5-Fu bodies is obstructed, and the half-life significantly extends, and decomposes and weakens and synthesize
Increase, cytotoxicity also accordingly strengthens.FDA suggestions carry out the detection of DPYD gene pleiomorphisms before 5-Fu medicines are taken.
MTHFR reduction 5,10-CH2-THFAs are 5-methyltetrahydrofolate, and the former is the important original of thymidylic acid synthesis
One of material, participates in the synthesis of DNA and repairs；The latter is methyl donor main in vivo, participates in DNA methylation.Mthfr gene
The mutation of 677C → T makes 223 amino acids be changed into L-Valine → MTHFR enzymatic activity reduction → 5,10-MTHFR concentration from alanine
Improve：5-FdUMP and 5,10-MTHFR, TS form stable covalent complex, disturb the synthesis and reparation of DNA, improve 5-FU
Antitumous effect.
4th, the personalized medicine related gene of vincristine
Vinca medicine can disturb the formation of proliferative cell spindle, make mitosiss stop at mid-term, at higher dose
Under amount, chromosome can be directly destroyed, microtubular protein crystallization is made, prevents which from assembling.For cell cycle specific agents, effect
In the M phases, and there is retarding action to the M phases, its markedly fast, catabasises are short.Additionally, vinca medicine also has immunosuppressive action.
In vitro study has shown that vinca medicine is the substrate of ABCB1.P- glycoproteins (P-glycopretein, P-gp)
1 transport protein of box B subfamilies (ATP-binding cassette subfamily B member 1, ABCB1) is combined for ATP
The transmembrane transporter P-gp of gene code, the cross-film medicine output pump function with Energy Dependence, can be by intracellular substrate bag
Including various antitumor drug and pumping out extracellular makes intracellular drug accumulation lowering of concentration.The P-gp encoded by multidrug resistance gene (MDR1)
High expression, cause the main mechanism that drug efflux increase is that multidrug resistance occurs.Pharmacogenomicses and pharmacogeneticses grind
Study carefully expression or the activity for showing that MDR1 gene pleiomorphisms can change P-gp, and then cause multidrug resistance.On ABCB1 genes
1199th, the change in 2677 and 3,435 3 sites can cause the mRNA of the gene to express reduces, so as to reduce drug efflux pump
Go out the speed of cell, increased the sensitivity of medicine, reduce drug resistance.
5th, the personalized medicine related gene of paclitaxel
Taxanes are a kind of new breast cancers, and tubulin binding, by promoting cell between its division
Phase forms atypical micro-pipe framework and prevents the normal physiological depolymerization of micro-pipe, makes the tumor cell of quick division have silk point
Split in the phase and be firmly limited in this framework, it is finally dead because duplication is blocked.For cell cycle specific agents, master
Act on G2 late periods and M phases.Antitumor spectra is wide, and therapeutic index is high, and has radiosensitizing effect.
Transhipment substrate of the Taxane family for P- glycoproteins (P-glycoprotein, P-gp).The gene pleiomorphism of ABCB1
Its phenotypic genetic factor can be affected, it is also possible to take part in the regulation and control of ABCB1 expression.P- glycoprotein (P-glycopretein, P-
Gp) for ATP combine 1 transport protein of box B subfamilies (ATP-binding cassette subfamily B member 1,
ABCB1) the transmembrane transporter P-gp of gene code, the cross-film medicine output pump function with Energy Dependence can will be intracellular
Substrate includes that various antitumor drug are pumped out extracellular makes intracellular drug accumulation lowering of concentration.On 1236 sites on MDR1 genes
TT types in TT types and 3435 sites can reduce the mRNA expression of the gene, reduce medication effect.
6th, the personalized medicine related gene of tamoxifen
Tamoxifen is a kind of selective estrogen receptor modulatorss, is usually used in the auxiliary treatment of breast carcinoma, it is possible to decrease
47% recurrence and 26% mortality rate.Tamoxifen by CYP450 enzymes be metabolized as active N- demethyls tamoxifen and 4- hydroxyls he
Former times is not fragrant, and 4- hydroxyls tamoxifen is further metabolized as activated product 4- hydroxyl-N- demethyl tamoxifens by CYP2D6
(Endoxifen), the latter and estrogen receptor affinity are high, suppress estrogen-dependent breast cancer cell multiplication ability be he not
30-100 times of former times sweet smell.
When CYP2D6 genovariations cause the decline of CYP2D6 enzymatic activitys or enzymatic activity to be suppressed, activated product in blood plasma
Endoxifen will be decreased obviously.Research shows that the gene of CYP2D6 has polymorphism, and polymorphism causes the CYP2D6 enzymes for encoding
Activity produces difference.The curative effect of patient's tamoxifen of the weak metabolism of CYP2D6 is reduced.And CYP2D6*2A is increased with tamoxifen curative effect
It is strong relevant.
7th, the personalized medicine related gene of irinotecan
Irinotecan (CPT-11) is semisynthetic camptothecin soluble derivative, can be hydrolyzed to rapidly in liver
The metabolite SN-38 of activity, the latter are typeⅠtopoisomerase inhibitor, repair, disturb answering for DNA after can suppressing DNA single-strand breaks
System and transcription, cause death of neoplastic cells, and active metabolite SN-38 is also the basis for producing Graft Versus Tumor.The metabolism of SN-38
UGT1A1 inactivations mainly in Jing uridine diphosphate glucuronatetransferases family, particularly liver are produced for glucuronic acid
Thing (SN-38G), then Jing bile excretions enter intestinal, intestinal bacteria GRD beta-glucuronidase effect under be converted to SN-38, draw
Send out intestinal mucosa injury；And the UGT1A1 in intestinal can be catalyzed SN-38 once again for SN-38G, releasing intestinal mucosal injury.
Research finds (the 1st exon 2 11) heterozygous mutant in the patient treated using irinotecan by Aisan
It is thirsty that G/A and homozygous mutant A/A dramatically increase patient generation 3/4 grade of Neutrophilic granulocytopenia, thrombocytopenia and Delayed onset abdomen
Risk.The expression of UGTs enzymes and its activity are closely related with the curative effect of CPT-11 and untoward reaction.
8th, the personalized medicine related gene of cyclophosphamide
Cyclophosphamide (Cyclophophamide, CPA) is clinical conventional chemotherapeutics, itself no cytotoxicity and
Immunosuppressive action, the effect of Jing cytochrome P 450 enzymes (CYP450) metabolism competence exertion, generation between Different Individual after entering in vivo
Thank to speed difference very big.CPA Jing CYPs are metabolized as active metabolite 4- hydroxyl cyclophosphamide (4-
Hydroxycyclophoplaamide, 4-OH CPA), the latter is further broken into the phosphorus of therapeutic effect in nucleus
Amide chlormethine, the acrylic aldehyde without therapeutic effect.
The S- mephenytoin hydroxylases of CYP2C19 gene codes, are one of members of main enzyme system of drug disposition metabolism.
CYP2C19*1 is wild type, and S- mephenytoins hydroxylase is normal enzymatic activity, and CYP2C19*2 is 681G>A, S- mephenytoin hydroxyl
Changing enzyme reduces for enzymatic activity, and CYP2C19*3 is 636G>A, S- mephenytoin hydroxylase is reduced for enzymatic activity, the two mutation positions
Point accounts for more than the 99% of the weak metabolic phenotype of Asians.Therefore FDA suggestions pharmacist can be by detecting the genotype of CYP2C19 come next
Understand metabolic capacity of the patient to above medicine, weak metabolic patient can select other corresponding medicines for more than.XRCC1
BER (base excision repair approach) and the singly-bound fracture restoration caused because of ionizing radiation and oxidative damage is participated in, to maintaining gene
Stability play pivotal role.On the gene, the SNP of A1196G is substantially related to chemotherapy effect.
9th, the personalized medicine related gene of methotrexate
Tetrahydrofolic acid be in vivo purine biosynthesis nucleotide and fudr acid important coenzyme, methotrexate conduct
A kind of folic acid reductase inhibitor, it is main to suppress dihydrofolate reductase and prevent dihydrofoilic acid from being reduced into physiologically active
Tetrahydrofolic acid, so that the transferance of a carbon-based group is obstructed in the biosynthetic process of purine nucleotides and pyrimidine nucleotide,
The biosynthesiss of DNA are caused to be suppressed.Additionally, methotrexate also has the inhibitory action to thymus nucleoside acid enzyme, but press down
RNA processed is then weaker with the effect of protein synthesis, mainly acts on the S phases of cell cycle, belongs to cell cycle specific agents, right
The cell of G1/S phases also has retarding action, and the effect to G1 phase cells is weaker.
Methotrexate action target is that MTHFR. can block dihydrofoilic acid and be changed into tetrahydrofolic acid, so as to suppress thymus pyrimidine
Nucleotide synthesizes.Its main SNP site is that the usual MTHFR C667T loci polymorphisms of C667T and A1298C. make enzyme activity respectively
Reducing MTHFR C667T mutation can make homocysteine be converted into methionine reduction, make homocysteine level in blood
Raise, cause intracellular folate cycle disorderly, cause the reactivity and toxicity of MTX to raise (mainly toxicity rising).With
C667C wild-type homozygote patients compare .T667T saltant type homozygote patient MTHFR enzyme activity reduces by 70%, but TT type patient's blood
Paddle acid concentration is lower than CC and CT type patients.Serious oral mucositis and other serious bloods that TT genotype is caused with MTX
Toxic reaction is relevant.The single nucleotide polymorphism of ABCB1 genes has been demonstrated related to the expression of P- glycoproteins, and one of them is
The 26th exon 3 435C of the gene>T polymorphisms.Some researchs show that the higher expression of P- glycoproteins may be methotrexate
The mark of drug resistance.
10th, the personalized medicine related gene of letrozole, Anastrozole
Arimedex is very important class medicine in current endocrinotherapy for breast cancer.This class medicine can
To be combined with aromatase, it is allowed to lose the activity of enzyme, so that androgen cannot be converted into estrogen, cut-out elderly woman is female
The source of hormone, reaches the purpose for the treatment of estrogen receptor positive breast carcinoma.Exemestane, letrozole, Anastrozole are current
3rd generation arimedex of wide clinical application.
CYP19A1 polymorphisms are altered by estrogen level and tumor characteristic, so as to affect arimedex
Therapeutic effect, and affect patient with breast cancer's prognosis.To postmenopausal estrogen receptor positive metastatic breast cancer patient using virtue
The association study discovery of the therapeutic effect of sweetening treatment enzyme inhibitor letrozole and the single nucleotide polymorphism of CYP19A1, primary care
Disease developing time (TTP) carry G161T mutant genes patient's body in be obviously prolonged.
11st, the personalized medicine related gene of epirubicin
Epirubicin belongs to cell cycle nonspecific agent (CCNSA), and Main Function position is nucleus, can be directly embedded into DNA
Between base pair, transcription is disturbed, prevent the formation of mRNA, so as to suppress the synthesis of DNA and RNA.To Topoisomerase
There is inhibitory action.Epirubicin is the isomerss of amycin, and compared with amycin, chemotherapeutic index is high, and anticancer therapeutic is equal
Or it is slightly higher, but the toxicity of heart is less.Clinically it is used for acute leukemia and malignant lymphoma, breast carcinoma, lung bronchogenic carcinoma, ovum
Nest cancer, nephroblastoma, soft tissue sarcoma, bladder cancer, carcinoma of testis, carcinoma of prostate, gastric cancer, hepatocarcinoma (include primary liver cell
Cancer and metastatic carcinoma) and various solid tumors such as medullary thyroid carcinoma.
Glutathione S-transferase (GSTP1) is important detoxication enzyme family member, and its major function is catalyzed in certain
The electrophilic group of source property or external harmful substance is coupled with the sulfydryl of reduced glutathion, is increased its hydrophobicity and is made it easier to
Cell membrane is passed through, and is excreted after being decomposed, prevent body DNA damage.Can also say potential in vivo by non-enzymatic binding mode
Cytotoxic chemical medicine is discharged from vivo, so as to reach the purpose of removing toxic substances.Research display, SNP and the epirubicin of GSTP1 genes
Toxicity is closely related.
12nd, the personalized medicine related gene of Capecitabine
Capecitabine is a kind of antimetabolic fluoropyrimidine deoxynucleoside carbamates medicine that can be transformed into 5-FU in vivo
Thing, trade name xeloda can suppress cell division, RNA interfering and protein synthesis.It is mainly used in advanced primary or turns
The treatment of shifting property breast carcinoma, rectal cancer, colon cancer and gastric cancer.
DPYD gene code dihydropyrimidine dehydrogenases, Main Function be decompose cell in useless uracil and thymus it is phonetic
Pyridine, is separately converted to 5,6- dihydrouracil and 5,6- dihydrothymine.Due to tumour medicine such as 5-FU and Ka Peita
With the structure similar with pyrimidine, dihydropyrimidine dehydrogenase can equally decompose such medicine, and DPYD genes is polymorphic for shore etc.
Property affect enzymatic activity, therefore, DPYD gene pleiomorphisms are closely related with the drug reaction of Capecitabine.
In recent years, with the development of Molecular Biology and technology, chemotherapy of tumors medication related mechanism and medication are related
Molecular basises major part be elucidated with, the method for establishing quick detection chemotherapy of tumors personalized medicine related gene, is swollen
Tumor chemotherapy quick medicament sensitivity testss open up a new way.
But current molecular detecting method is mainly sequenced with restriction fragment length polymorphism analysis, PCR, gene chip is
It is main, the defect such as generally existing small throughput, low resolution, high background signal, high cost.
MassArray molecular weight array techniques are gene mutation analysis technologies leading in the world.MassArray is combined
Simply, reliable primer extension reaction and advanced MALDI-TOF mass-spectrometric techniques, can carry out genotype point with fast, economical
Analysis, reaches highest accuracy rate and cost performance in the market.IPLEX GOLD based on MassArray molecular weight array Platforms
Technology can easily design up to 40 kinds of genotype detection.Experimental design is flexibly, cheap.
The content of the invention
It is an object of the present invention to provide a kind of method of detection chemotherapy of tumors personalized medicine related gene.
It is a further object to provide a kind of test kit of detection chemotherapy of tumors personalized medicine related gene.
According to an aspect of the invention, there is provided a kind of method of detection chemotherapy of tumors personalized medicine related gene.
Wherein, the chemotherapy of tumors personalized medicine related gene is included selected from UGT1A1-G211A, MTHFR-C677T, MDR1-
G2677T/A、XRCC1-A1196G、MDR1-G1199A、CYP2C19-G681A、CDA-G208A、CYP2C9-A1075C、
CYP2D6-C100T、XRCC1-C27157T、CYP2C19-G636A、CYP19A1-G161T、MDR1-C3435T、GSTP1-
One or more in A313G, UGT1A1-C686A, MDR1-C1236T, DPYD-G1905+1A and CDA-A79G/C；It is preferred that
Ground, the chemotherapy of tumors personalized medicine related gene include UGT1A1-G211A, MTHFR-C677T, MDR1-G2677T/A,
XRCC1-A1196G、MDR1-G1199A、CYP2C19-G681A、CDA-G208A、CYP2C9-A1075C、CYP2D6-C100T、
XRCC1-C27157T、CYP2C19-G636A、CYP19A1-G161T、MDR1-C3435T、GSTP1-A313G、UGT1A1-
C686A, MDR1-C1236T, DPYD-G1905+1A and CDA-A79G/C；It is highly preferred that the chemotherapy of tumors personalized medicine phase
Correlation gene by UGT1A1-G211A, MTHFR-C677T, MDR1-G2677T/A, XRCC1-A1196G, MDR1-G1199A,
CYP2C19-G681A、CDA-G208A、CYP2C9-A1075C、CYP2D6-C100T、XRCC1-C27157T、CYP2C19-
G636A、CYP19A1-G161T、MDR1-C3435T、GSTP1-A313G、UGT1A1-C686A、MDR1-C1236T、DPYD-
G1905+1A and CDA-A79G/C compositions.
Methods described includes：
1) preparation of pcr amplification primer thing：
Pcr amplification primer thing is according to the special of selected chemotherapy of tumors personalized medicine related gene design to be detected
Property for every kind of personalized medicine related gene amplimer, the amplimer it is amplifiable including including mutational site one
Segment DNA sequence.The amplimer (including forward primer and reverse primer) is at 3' ends with the gene order targeted with which
The 17-25 base for matching completely, has sequence label at 5' ends, preferably has identical sequence label at 5' ends, more preferably
The sequence label is ACGTTGGATG (SEQ ID NO:55), distinguishing amplimer and extension primer.
Preferably, amplimer of the invention can be included selected from SEQ ID NO:1～SEQ ID NO:A pair in 36
Or it is multipair；It is highly preferred that the amplimer of the present invention can include SEQ ID NO:1～SEQ ID NO:36；And most preferably,
The amplimer of the present invention can be by SEQ ID NO:1～SEQ ID NO:36 compositions.
2) preparation of mass spectrum extension primer：
The length of mass spectrum extension primer is for 15-28 base and its 3' end is located at personalized medicine associated gene mutation position
A upper base in the direction of extension of point, extension primer extend a base, the alkali of extension when extension occurs, only
Base is personalized medicine associated gene mutation site.
Wherein, the molecular weight difference between extension products and extension primer is not less than 9Da；According between primer, primer with expand
Dimer is not formed between volume increase thing/extension products, primer itself is not formed
The principle design primer matched somebody with somebody.Especially, the extension primer for Mass Spectrometer Method is designed according to following principle：Each extension products point
The mutual difference of son amount is not less than 30Da, and the molecular weight difference between each extension primer and each extension products is not less than 9Da, prolongs
Extend thing and extension products molecular weight between 4500-8500Da.
Preferably, extension primer of the invention can be included selected from SEQ ID NO:37～SEQ ID NO:One in 54
Or it is multiple；It is highly preferred that the extension primer of the present invention can include SEQ ID NO:37～SEQ ID NO:54；And most preferably
Ground, the extension primer of the present invention can be by SEQ ID NO:37～SEQ ID NO:54 compositions.
3) preparation of detection template：Extract sample to be tested DNA.
Sample DNA is extracted using appropriate commercial kit, clinical sample is tried using Tiangen extracting genome DNA
Agent box.
4) with step 3) in extract DNA as template, using step 1) in pcr amplification primer thing by PCR amplification, acquisition
Target sequence amplification product.
5) by SAP ferment treatments, the unreacted dNTP contained in the amplified production that 4) removing step obtains.
6) with step 5) in the amplified production that obtains as template, using step 2) in mass spectrum extension primer by extending instead
Should, in 3 ' one base of end connection of extension primer, so as to obtain extension products.
7) purification step 6) in obtain extension products.
8) product after purification is carried out Mass Spectrometer Method on mass spectrograph.Resulting mass spectrum peak figure is entered by analysis software
Row analysis, obtains genotyping result.
According to another aspect of the present invention, there is provided a kind of reagent of detection chemotherapy of tumors personalized medicine related gene
Box, the test kit include：
1) pcr amplification primer thing：
Pcr amplification primer thing is according to the special of selected chemotherapy of tumors personalized medicine related gene design to be detected
Property for every kind of personalized medicine related gene amplimer, the amplimer it is amplifiable including including mutational site one
Segment DNA sequence.The amplimer (including forward primer and reverse primer) is at 3' ends with the gene order targeted with which
The 17-25 base for matching completely, has sequence label at 5' ends, preferably has identical sequence label at 5' ends, more preferably
The sequence label is ACGTTGGATG (SEQ ID NO:55), distinguishing amplimer and extension primer.
Preferably, amplimer of the invention can be included selected from SEQ ID NO:1～SEQ ID NO:A pair in 36
Or it is multipair；It is highly preferred that the amplimer of the present invention can include SEQ ID NO:1～SEQ ID NO:36；And most preferably,
The amplimer of the present invention can be by SEQ ID NO:1～SEQ ID NO:36 compositions.
2) mass spectrum extension primer：
The length of mass spectrum extension primer is for 15-28 base and its 3' end is located at personalized medicine associated gene mutation position
A upper base in the direction of extension of point, extension primer extend a base, the alkali of extension when extension occurs, only
Base is personalized medicine associated gene mutation site.
Preferably, extension primer of the invention can be included selected from SEQ ID NO:37～SEQ ID NO:One in 54
Or it is multiple；It is highly preferred that the extension primer of the present invention can include SEQ ID NO:37～SEQ ID NO:54；And most preferably
Ground, the extension primer of the present invention can be by SEQ ID NO:37～SEQ ID NO:54 compositions.
Preferably, the test kit of detection chemotherapy of tumors personalized medicine related gene of the invention includes：
1) PCR reactant liquors Mix：Including 1 × PCR reaction buffers (being purchased from Agena Bioscience companies), above-mentioned PCR
Amplimer (gives birth to work biosynthesiss by Shanghai)；
2) PCR reactions Taq enzyme (being purchased from Agena Bioscience companies)；
3) SAP reaction buffers (being purchased from Agena Bioscience companies)；
4) SAP enzymes (being purchased from Agena Bioscience companies)；
5) Extension reactant liquors Mix：Including reaction buffer (being purchased from Agena Bioscience companies), above-mentioned matter
Spectrum extension primer (gives birth to work biosynthesiss by Shanghai)；
6) Extension reactions Taq enzyme (being purchased from Agena Bioscience companies)；
7) MassARRAY chips (being purchased from Agena Bioscience companies).
Compared with prior art, the present invention has advantages below：
1) the reagent consumptive material for using is relatively easy and stable, it is not necessary to the expensive examination such as fluorescent dye, special enzyme
Agent；
2) reaction can be carried out in micro system, reduce the use of sample and various consumable goodss；
3) due to mass-spectrometric technique direct detection DNA molecular weight (mass-to-charge ratio) and directly determine base type (that is, be not required to
To change through any type of signal), therefore, as long as there is the amplification of DNA fragments of a copy to know by being amplified in theory
Not, so as to high sensitivity；
4) the features such as mass-spectrometric technique also has automatization, high throughput testing, therefore, mass-spectrometric technique and many primer extension technique phases
With reference to simultaneously can detecting multiple medication related locus in a reaction system, so as to greatly reduce workload, improve
Detection flux.
Especially, technical scheme adopts flight time mass spectrum genotyping system, a set of brand-new by designing
Mass spectrum extension primer, realize detection and the typing of the personalized medicine related gene to said medicine.Which is existing with other
Method is compared, and can accurately detect chemotherapy of tumors personalized medicine dependency simultaneously, with high flux, automatization, low cost
The characteristics of, there is significant advantage.
Description of the drawings
Fig. 1 is the mass spectra peak detected to CYP2C19-G681A with the DNA of normal clinical sample 3-A extractions as template
The figure of map analysis result, its result are homozygosis G, show low to Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk.
Fig. 2 be the DNA extracted with the tumor tissues clinical sample 2-A of tumor patient as template, CYP2C19-G681A is entered
The figure of the mass spectra peak map analysis result of row detection, its result are heterozygosis GA, show and Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk is fitted
In, curative effect is moderate.
Fig. 3 be the DNA extracted with the tumor tissues clinical sample 2-B of tumor patient as template, CYP2C19-G681A is entered
Row detection mass spectra peak map analysis result figure, its result be homozygosis A, show to Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk compared with
Difference, curative effect are poor.
Fig. 4 is the mass spectra peak detected to CYP2C19-G681A with the DNA of normal clinical sample 4-A extractions as template
The figure of map analysis result, its result are homozygosis G, show low to Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk.
Fig. 5 is the mass spectra peak detected to CYP2C19-G681A with the DNA of normal clinical sample 5-A extractions as template
The figure of map analysis result, its result are homozygosis G, show low to Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk.
Fig. 6 is the mass spectra peak detected to CYP2C19-G681A with the DNA of normal clinical sample 6-A extractions as template
The figure of map analysis result, its result are homozygosis G, show low to Treated with Chemotherapy with Cyclophosphamide medicine poison pair risk.
Specific embodiment
With reference to specific embodiment, the invention will be further described, but the present invention is not limited to following examples.
In following embodiments, if no special instructions, it is conventional method.
Primer sequence by Shanghai give birth to work biosynthesiss, PCR amplifing reagents, SAP enzymes, Extension reactant liquors,
MassARRAY chips are purchased from Agena Bioscience companies, and PCR amplification instrument is 9700 model of AB companies, mass spectrometer
For Agena Bioscience companies MassARRAY Compact Analyzer.
Embodiment
The design of 1 primer of embodiment
(1) pcr amplification primer thing：
According to the personalized medicine related gene of selected chemotherapy of tumors individuation medicine to be detected, design special
Property for every kind of personalized medicine related gene amplimer, amplifiable a section including including mutational site of amplimer
DNA sequence.At least 15 bases that the amplimer is matched with the gene order targeted with which completely at 3' ends, in 5'
Sequence label of the end with 10 bases (acgttggatg), to distinguish amplimer and extension primer.
(2) mass spectrum extension primer：
Design extension primer, the length of the extension primer is 15-28 base, and its 3' end is related prominent positioned at medication
Become a upper base in the direction of extension in site, extension primer extends a base when extension occurs, only, extends
Base be medication relevant mutational site.Wherein, between the wild type extension products and saltant type extension products of extension primer
Molecular weight difference be not less than 9Da (Da=dalton), and the molecular weight between the extension products of each medication relevant mutational site
Difference is not less than 30Da.According to dimer, primer itself is not formed between primer, between primer and amplified production/extension products not
Form the principle design primer that mispairing does not occur between hairpin structure and primer and template.Especially, prolonging for Mass Spectrometer Method
The thing that extends is designed according to following principle：The mutual difference of the molecular weight of each extension products is not less than 30Da, each extension primer with
Molecular weight difference between each extension products is not less than the molecular weight of 9Da, extension primer and extension products in 4300-8500Da
Between.
It is as shown in table 1 for described personalized medicine associated gene mutation site information that the present invention is designed, and amplification is drawn
, referring to table 2, extension primer is referring to table 3 for thing.
Table 1：Personalized medicine associated gene mutation information
Table 2：For the pcr amplification primer thing sequence of PCR amplifications
Table 3：For the mass spectrum extension primer of the extension
The preparation of 2 detection template of embodiment
Sudden change sample is tumor tissues (numbering be 2-A and 2-B) and health adult tissue sample of the hospital by 2 tumor patients of inspection
This 4 parts (numbering is 3-A, 4-A, 5-A, 6-A) is extracted DNA and (is extracted using Tiangen DNA test kits, operational approach is according to examination
Agent box description is extracted).
The detection method of 3 chemotherapy of tumors personalized medicine related gene of embodiment
(1) DNA extracted with embodiment 2 is expanded by PCR as template using the pcr amplification primer thing in embodiment 1,
Obtain target sequence amplification product.Pcr amplification reaction system is referring to table 4.Wherein, all reagents are bought from Agena Bioscience
Company.
Table 4：Pcr amplification reaction system
PCR reaction conditions are 94 DEG C, 2 minutes；94 DEG C of degeneration 30 seconds, 56 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, expand altogether
Increase 45 to circulate；Final 72 DEG C extend 5 minutes.In the present embodiment, expanded as PCR using the DNA extracted in embodiment 2
DNA profiling.Meanwhile, using aseptic double-distilled water as negative control.By check sample with sample to be tested according to identical course of reaction
Reacted and tested, to verify the effectiveness of detection.
(2) by SAP ferment treatments, the unreacted dNTP contained in the amplified production that removing step (1) is obtained.SAP enzymes
Reaction system is shown in Table 5.All reagents are bought from Agena Bioscience companies.
Table 5：SAP enzyme reaction systems
| Reagent | Volume/reaction |
| Water (HPLE levels) | 1.53μl |
| SAP enzyme buffer liquids | 0.17μl |
| SAP enzymes | 0.30μl |
| Pcr amplification product is obtained in step (1) | 5.0μl |
| Cumulative volume | 7.0μl |
SAP enzyme reactions condition is 37 DEG C and incubates 40 minutes, to remove remaining dNTP in pcr amplification reaction；85 DEG C incubate 5
Minute, so that SAP enzymes inactivation.
(3) amplified production obtained with (2) is as template, anti-by extending using the mass spectrum extension primer in embodiment 1
Should, in 3 ' one base of end connection of extension primer, so as to obtain extension products.Extension system is shown in Table 6.All reagent purchases
Buy from Agena Bioscience companies.
Table 6：Extension system
* wherein extension primer mixture carries out linear relationship adjustment (that is, according to every kind of according to the molecular size range of each primer
The usage amount of the every kind of primer of molecular weight calculation of extension primer).
Extension condition is 94 DEG C, 30 seconds；94 DEG C of degeneration 5 seconds, 52 DEG C are annealed 5 seconds, and 80 DEG C extend 5 seconds, coamplification 40
Individual circulation, and annealing and extension carry out 5 partial circulatings in each cycle；Final 72 DEG C extend 3 minutes.
(4) using the extension products obtained in resin (purchase is from Agena Bioscience companies) purification step (3).
6mg resins, 16.00 μ l water is added vertically to shake up one hour in extension products.After the reaction of this step, resin will be with reactant
Cation in system is fully combined, so that reaction system desalination.Purified product after the completion of reaction can preserve a couple of days at 4 DEG C,
Also several weeks can be preserved at -20 DEG C.The purified product of gained after 4000rpm is centrifuged 5 minutes takes supernatant and is directly used in mass spectrum inspection
Survey.
(5) product after purification is carried out into Mass Spectrometer Method on Agena Bioscience MALDI-TOF mass spectrographs.Institute
The mass spectrum peak figure for obtaining is analyzed by Typer4.0 analysis softwares (being provided by Agena Bioscience companies), is divided
Type result.
Testing result：
Mass spectra peak map analysis result is as shown in figs 1 to 6.
Claims (10)
1. it is a kind of detection chemotherapy of tumors personalized medicine related gene method, wherein, the chemotherapy of tumors personalized medicine phase
Correlation gene is included selected from UGT1A1-G211A, MTHFR-C677T, MDR1-G2677T/A, XRCC1-A1196G, MDR1-
G1199A、CYP2C19-G681A、CDA-G208A、CYP2C9-A1075C、CYP2D6-C100T、XRCC1-C27157T、
CYP2C19-G636A、CYP19A1-G161T、MDR1-C3435T、GSTP1-A313G、UGT1A1-C686A、MDR1-C1236T、
One or more in DPYD-G1905+1A and CDA-A79G/C；Preferably, the chemotherapy of tumors personalized medicine related gene
Including UGT1A1-G211A, MTHFR-C677T, MDR1-G2677T/A, XRCC1-A1196G, MDR1-G1199A, CYP2C19-
G681A、CDA-G208A、CYP2C9-A1075C、CYP2D6-C100T、XRCC1-C27157T、CYP2C19-G636A、
CYP19A1-G161T、MDR1-C3435T、GSTP1-A313G、UGT1A1-C686A、MDR1-C1236T、DPYD-G1905+1A
And CDA-A79G/C；It is highly preferred that the chemotherapy of tumors personalized medicine related gene is by UGT1A1-G211A, MTHFR-
C677T、MDR1-G2677T/A、XRCC1-A1196G、MDR1-G1199A、CYP2C19-G681A、CDA-G208A、CYP2C9-
A1075C、CYP2D6-C100T、XRCC1-C27157T、CYP2C19-G636A、CYP19A1-G161T、MDR1-C3435T、
GSTP1-A313G, UGT1A1-C686A, MDR1-C1236T, DPYD-G1905+1A and CDA-A79G/C are constituted,
Methods described includes：
1) preparation of pcr amplification primer thing：Pcr amplification primer thing is to be set according to selected personalized medicine related gene to be detected
Amplimer of the specificity of meter for every kind of gene, the amplifiable section of DNA including including mutational site of the amplimer
Sequence, the 17-25 base that the amplimer is matched with the gene order targeted with which completely at 3' ends have at 5' ends
There is sequence label, to distinguish amplimer and extension primer；
2) preparation of mass spectrum extension primer：It is individual that the length of the mass spectrum extension primer is that 15-28 base and its 3' end are located at
A upper base in the direction of extension in body medication associated gene mutation site, extension primer occur extension when,
Only extend a base, the base of extension is personalized medicine associated gene mutation site；
3) preparation of detection template：Extract sample to be tested DNA；
4) with step 3) in extract DNA as template, using step 1) in the pcr amplification primer thing by PCR amplification, acquisition
Target sequence amplification product；
5) by SAP ferment treatments, the unreacted dNTP contained in the amplified production that 4) removing step obtains；
6) with step 5) in the amplified production that obtains as template, using step 2) in the mass spectrum extension primer by extending instead
Should, in 3 ' one base of end connection of extension primer, so as to obtain extension products；
7) purification step 6) in obtain extension products；
8) product after purification carries out on mass spectrograph Mass Spectrometer Method, resulting mass spectrum peak figure is carried out point by analysis software
Analysis, obtains genotyping result.
2. method according to claim 1, wherein, step 1) in, the amplimer has identical label at 5' ends
Sequence.
3. according to method in any one of the preceding claims wherein, wherein, step 1) in, the sequence label is
ACGTTGGATG(SEQ ID NO:55)。
4. according to method in any one of the preceding claims wherein, wherein, step 1) in, the amplimer includes being selected from
SEQ ID NO:1～SEQ ID NO:It is one or more pairs of in 36；Preferably, the amplimer includes SEQ ID NO:1～
SEQ ID NO:36；It is highly preferred that the amplimer is by SEQ ID NO:1～SEQ ID NO:36 compositions.
5. according to method in any one of the preceding claims wherein, wherein, step 2) in, the extension primer includes being selected from
SEQ ID NO:37～SEQ ID NO:One or more in 54；Preferably, the extension primer includes SEQ ID NO:37
～SEQ ID NO:54；It is highly preferred that the extension primer is by SEQ ID NO:37～SEQ ID NO:54 compositions.
6. a kind of test kit of detection chemotherapy of tumors personalized medicine related gene, the test kit includes：
1) pcr amplification primer thing：Pcr amplification primer thing is the spy according to selected personalized medicine related gene design to be detected
Amplimer of the opposite sex for every kind of gene, the amplifiable section of DNA sequence including including mutational site of the amplimer, institute
The 17-25 base that amplimer is matched with the gene order targeted with which completely at 3' ends is stated, there is label at 5' ends
Sequence, to distinguish amplimer and extension primer；
2) mass spectrum extension primer：The length of the mass spectrum extension primer is that 15-28 base and its 3' end are used positioned at individuation
A upper base in the direction of extension in medicine phases correlation gene mutational site, extension primer only extend when there is extension
One base, the base of extension are personalized medicine associated gene mutation site.
7. test kit according to claim 6, wherein, the amplimer has identical sequence label at 5' ends.
8. the test kit according to claim 6 or 7, wherein, step 1) in, the sequence label is ACGTTGGATG (SEQ
ID NO:55)。
9. the test kit according to any one of claim 6-8, wherein, the amplimer is included selected from SEQ ID NO:
1～SEQ ID NO:It is one or more pairs of in 36；Preferably, the amplimer includes SEQ ID NO:1～SEQ ID NO:
36；It is highly preferred that the amplimer is by SEQ ID NO:1～SEQ ID NO:36 compositions.
10. the test kit according to any one of claim 6-9, wherein, the extension primer is included selected from SEQ ID
NO:37～SEQ ID NO:One or more in 54；Preferably, the extension primer includes SEQ ID NO:37～SEQ ID
NO:54；It is highly preferred that the extension primer is by SEQ ID NO:37～SEQ ID NO:54 compositions.
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