CN102312008A - Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments - Google Patents
Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments Download PDFInfo
- Publication number
- CN102312008A CN102312008A CN201110301725A CN201110301725A CN102312008A CN 102312008 A CN102312008 A CN 102312008A CN 201110301725 A CN201110301725 A CN 201110301725A CN 201110301725 A CN201110301725 A CN 201110301725A CN 102312008 A CN102312008 A CN 102312008A
- Authority
- CN
- China
- Prior art keywords
- seq
- gene
- sequence
- snp
- phase chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 239000007791 liquid phase Substances 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title abstract description 31
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title abstract description 21
- 239000003814 drug Substances 0.000 title abstract description 18
- 229910052697 platinum Inorganic materials 0.000 title abstract description 11
- 230000001225 therapeutic effect Effects 0.000 title abstract description 3
- 239000002773 nucleotide Substances 0.000 title description 2
- 125000003729 nucleotide group Chemical group 0.000 title description 2
- 230000000694 effects Effects 0.000 claims description 33
- 241000183024 Populus tremula Species 0.000 claims description 28
- 229960004562 carboplatin Drugs 0.000 claims description 21
- 101150105460 ERCC2 gene Proteins 0.000 claims description 16
- 101150050700 ERCC1 gene Proteins 0.000 claims description 12
- 101150042435 Xrcc1 gene Proteins 0.000 claims description 10
- 101150008380 gstp1 gene Proteins 0.000 claims description 8
- 238000013461 design Methods 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 3
- 190000008236 carboplatin Chemical compound 0.000 claims 9
- 239000004005 microsphere Substances 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 108700028369 Alleles Proteins 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 14
- YAYRGNWWLMLWJE-UHFFFAOYSA-L carboplatin Chemical compound O=C1O[Pt](N)(N)OC(=O)C11CCC1 YAYRGNWWLMLWJE-UHFFFAOYSA-L 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 description 11
- 102100035184 General transcription and DNA repair factor IIH helicase subunit XPD Human genes 0.000 description 11
- 102000002258 X-ray Repair Cross Complementing Protein 1 Human genes 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 description 9
- 101100226017 Dictyostelium discoideum repD gene Proteins 0.000 description 8
- 108010000443 X-ray Repair Cross Complementing Protein 1 Proteins 0.000 description 8
- 108700031763 Xeroderma Pigmentosum Group D Proteins 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 102100030943 Glutathione S-transferase P Human genes 0.000 description 7
- 101001010139 Homo sapiens Glutathione S-transferase P Proteins 0.000 description 7
- 230000000875 corresponding effect Effects 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000011518 platinum-based chemotherapy Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000007096 poisonous effect Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 101000829171 Hypocrea virens (strain Gv29-8 / FGSC 10586) Effector TSP1 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010033109 Ototoxicity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007056 liver toxicity Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000262 ototoxicity Toxicity 0.000 description 1
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
- 229960001756 oxaliplatin Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 150000003058 platinum compounds Chemical class 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses specific sequences and a liquid-phase chip for SNP detection of genes related to therapeutic effectiveness of platinum medicaments. The liquid-phase chip mainly comprises: wild and mutant specific ASPE (allele specific primer extension) primer pairs selected from SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, and/or SEQ ID NO.9 and SEQ ID NO.10; microspheres respectively coated with specific anti-tag sequences selected from SEQ ID No.11 to SEQ ID No.20; and primers for amplification of the target sequences of SNP sites. The detection liquid-phase chip and detection method of the invention have high detection sensitivity, enhanced signal to noise ratio, and more accurate and reliable detection results.
Description
The application number of original application: 200910040095.9, the applying date: 2009-6-9
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, specific sequence and liquid-phase chip that the concrete SNP that relates to gene associated with treatment effect of carboplatin ERCC1, ERCC2, XRCC1 and GSTP1 detects.
Background technology
Since the antitumour activity of cis-platinum in 1967 came to light, the research of platinum-containing anticancer drug and application had obtained development rapidly.Developed at present and be the s-generation of representative and be the third generation platinum medicine of representative with the oxaliplatin with the carboplatin.The platinum class is a broad-spectrum anti-tumor medicine commonly used at present, has become indispensable medicine in the cancer chemotherapy.Though the curative effect of platinum medicine extensively approved, the difference between individuals of drug effect is very big and toxic side effect in various degree arranged after patient's medication.Its spinoff mainly is a Digestive tract toxicity, and common also can have renal toxicity, liver toxicity and bone marrow depression for feeling sick, vomitting, and ototoxicity and neurotoxicity are lighter.
The antitumor action of platinum medicine is through acting on DNA, forms the Pt-DNA binding substances, causes in the chain of DNA and interchain linkage, thereby suppresses the synthetic of DNA and duplicate.Vast amount of clinical is verified, and the SNP (SNP) of four genes is in close relations in the curative effect/toxic side effect of platinum medicine and the patient's body, i.e. ERCC1, ERCC2, XRCC1 and GSTP1.ERCC1 and ERCC2 are the key genes that the DNA nucleotide excision is repaired approach, and XRCC1 is the important factor in the DNA base excision reparation approach.GSTP1 is a kind of glutathione s-transferase, and it combines with gsh through the electrophilic group with toxic substance, plays the effect of protection cellular macromolecule.These four genes receive platinum compound destructive DNA to reply normal or make DNA avoid destruction through participating in physiological process such as DNA reparations, making, and in tumour cell, cause resistance, in normal cell, then can reduce toxic side effect.Therefore, the expert recommends the patient before accepting platinum-based chemotherapy, and the gene SNP of being correlated with detects, and helps the clinician to formulate the personalized medicine scheme according to patient's difference between individuals.To obviously improve curative effect of medication like this, reduce the generation of poisonous side effect of medicine.
A large amount of clinical studies show, the modal pleomorphism site that causes these four gene functions to weaken is respectively C19007T and the C8092A of ERCC1, the A2282C of ERCC2, the G1301A of XRCC1, and the A1578G of GSTP1.In Chinese population, the frequency of gene distribution of ERCC1-C19007T, ERCC1-C8092A, ERCC2-A2282C and XRCC1-G1301A is about 21%, 24%, 5% and 30% respectively; GSTP1-A1578G frequency of gene distribution about 21%.Big quantity research shows, in ERCC1, ERCC2, these three genes of XRCC1, carries the patient of or above miopragia allelotype, and when accepting platinum-based chemotherapy, the probability that toxic side effect takes place obviously raises, so that result of treatment is relatively poor.And the situation of GSTP1-A1578G gene polymorphism sites is just in time opposite, and genetic type is the patient of GA or GG, and is when accepting platinum-based chemotherapy, higher to the reactivity of platinum medicine, curative effect preferably arranged.Therefore,, judge whether to adopt platinum medicine or formulate the personalized medicine mode, can obviously improve curative effect of medication, reduce the generation of poisonous side effect of medicine according to patient's genotype through these five common function SNP sites are detected.
Having set up at present some is the technology of the detection gene mononucleotide polymorphism (SNP) on basis with PCR; Like direct sequencing, the sxemiquantitative round pcr, PCR-single-strand conformation polymorphism analysis (SSCP) detects; It is low that above technology has sensitivity, and sample is prone to shortcomings such as pollution, false positive rate height.Regular-PCR method and quantitative fluorescent PCR can not satisfy clinical needs owing to detect the limitation of flux.And polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analytical technology and once can only carry out the detection in a kind of SNP site, time and effort consuming based on the allelotrope discriminant analysis of TaqMan technology; Traditional solid phase chip costs an arm and a leg, and susceptibility is not high, and the repeatability of detected result is poor.Based on the principle of chip, it is the suspension liquid-phase chip technology of carrier with the microballoon that U.S. Luminex company has developed.This techniques make use polystyrene microsphere as detection platform, carries out high-throughout many index parallel detection to nucleic acid and protein and other with fluorescence detector as the carrier of reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the protein of different things to be detected or nucleic acid molecule as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and wherein red laser detects the red classification of microsphere surface intensity of fluorescence, and according to different color in the microballoon and number class, thereby confirm the type of reaction; Green laser detects the fluorescence intensity of fluorescent marker in the sample, detects microballoon kind, quantity through machine and computingmachine automatic statistical analysis laser again, thereby judges sample to be tested plurality of target tester concentration separately.Therefore, liquid-phase chip technology had both satisfied the requirement of high throughput testing, had possessed simultaneously quick and precisely, and was highly sensitive, and specificity is good, as a result advantage such as good reproducibility.We adopt the x-Taq liquid-phase chip technology can detect a plurality of SNP site simultaneously, realize the operation of fast and convenientization of high-throughput, have improved detection efficiency greatly, in similar detection technique, maintain the leading position.
Summary of the invention
One of the object of the invention provides and the closely-related ERCC1 of therapeutic effectiveness of platinum medicaments, ERCC2, XRCC1 and GSTP1 gene SNP detection liquid-phase chip.This liquid-phase chip can be used for 5 common SNP sites below individual event or the parallel detection: the C19007T of ERCC1 and C8092A, the A2282C of ERCC2, the G1301A of XRCC1, and the A1578G of GSTP1.
A kind of gene associated with treatment effect of carboplatin SNP detects liquid-phase chip, includes:
1. the wild-type and the special ASPE primer of mutant that design respectively to the SNP site of every kind of type are right; Every kind of ASPE primer is made up of the specific sequence that is directed against goal gene SNP site of 3 ' end and the tag sequence of 5 ' end, and said wild-type and mutant specificity ASPE primer are to being selected from respectively: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 and/or SEQ IDNO.9 and SEQ ID NO.10;
2. be coated with the microballoon of special anti-tag sequence 1 respectively, said anti-tag sequence can be correspondingly with (1) in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.11～SEQ ID NO.20; Preferably, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon;
3. to the amplimer of the target sequence in the A1578G SNP site of the G1301A of the A2282C of the C8092A of the C19007T of ERCC1 gene, ERCC1 gene, ERCC2 gene, XRCC1 gene and/or GSTP1 gene.Preferably; Amplify five primers respectively and be selected from the sequence among SEQ ID NO.21～SEQ ID NO.30 with the target sequence in SNP site; Promptly be directed against SEQ ID NO.21 and the SEQ ID NO.22 of C19007T, to SEQ ID NO.23 and the SEQ ID NO.24 of C8092A, to SEQ ID NO.25 and the SEQ ID NO.26 of A2282C; To SEQ IDNO.27 and the SEQ ID NO.28 of G1301A, to SEQ ID NO.29 and the SEQ ID NO.30 of A1578G.
Another object of the present invention provides and is used for the specific sequence that gene associated with treatment effect of carboplatin SNP detects, and this sequence characteristic is strong, can accurately distinguish the range gene type of SNP.
Said specific sequence is: to the SEQ ID NO.31 and the SEQ IDNO.32 in the SNP site of the C19007T of ERCC1 gene; SEQ ID NO.33 and SEQ ID NO.34 to the SNP site of the C8092A of ERCC1 gene; SEQ ID NO.35 and SEQ ID NO.36 to the SNP site of the A2282C of ERCC2 gene; SEQ ID NO.37 and SEQ ID NO.38 to the SNP site of the G1301A of XRCC1 gene; And/or to the SEQID NO.39 and the SEQ ID NO.40 in the SNP site of the A1578G of GSTP1 gene.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and PCR sequencing PCR is up to 100%.Prepared gene associated with treatment effect of carboplatin SNP detects liquid-phase chip and has extraordinary signal-NR, and does not have cross reaction basically between institute's designed probe and the anti-tag sequence.
2. designed ASPE type specificity primer of the present invention has extraordinary specificity, can accurately distinguish the range gene type of SNP.
3. use the detection method step of gene associated with treatment effect of carboplatin SNP detection liquid-phase chip of the present invention simple; Can accomplish five amplifications through a step multiplex PCR with the target sequence in SNP site; Many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided; Thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while.
4. the needed time of detection method provided by the present invention meets clinical needs especially well below sequencing technologies commonly used.
5. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and SNR strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 gene associated with treatment effect of carboplatin SNP detects liquid phase chip reagent box, mainly includes:
One, specific primer sequence (ASPE primer)
The respectively primer sequence of 5 common SNP sites difference designs specificity to gene associated with treatment effect of carboplatin.The ASPE primer is made up of the Tag+ specific primer sequence.The ASPE primer sequence is as shown in the table:
Table 1ASPE primer sequence (Tag+ special primer)
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (as shown in table 1) of wild-type.All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every ASPE primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon that encapsulates of anti-tag sequence
According to institute's designed ASPE Auele Specific Primer fragment; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE Auele Specific Primer fragment possibly form, as shown in table 2 with the anti-tag sequence on ten kinds of corresponding microballoons of tag sequence selecting:
Anti-tag sequence on table 2 microballoon corresponding with the Tag sequence of ASPE Auele Specific Primer right-hand member
Ten kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence encapsulate with microballoon on.Be connected with the spacerarm preface of 5-10 T between anti-tag sequence and the microballoon, promptly before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic anti-tag sequence is used sterilization ddH
2O is made into the stock solution of 100nmol/ml.The process that microballoon encapsulates is following:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, hatched 30 minutes, add the EDC working fluid of 2.5ul again, hatched again 30 minutes.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0), 1mmol/L EDTA] of 100ul, and 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of target sequence with SNP site:
C19007T, the C8092A of 5 kinds of common SNP site ERCC1 of target detect, the A2282C of ERCC2, the G1301A of XRCC1, and the A1578G of GSTP1 is on different gene or exon.Utilize Primer5.0 design five pairs of primers (seeing table 3), amplify five target sequences respectively with SNP site.
Table 3 amplifies the primer of the target sequence with SNP site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 utilization embodiment 1 described gene associated with treatment effect of carboplatin SNP detects the detection of liquid-phase chip to clinical sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5M?NaCl | Sigma?S5150 | 0.4M | 20ml |
| Triton?X-100 | Sigma?T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA. to be detected
Two, the pcr amplification of testing sample
Utilize five pairs of primers of Primer5.0 design; Multiplex PCR one step amplifies ERCC1 3 ' UTR and exon 4, ERCC2 exon 23, XRCC1 exons 10 and GSTP1 exon 5 totally five target sequences with SNP site, and the product size is respectively 638bp, 250bp, 507bp, 295bp and 399bp.Primer sequence (SEQ NO.21-30) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ NO.21-30 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.
Three, the enzyme of PCR product is cut processing
With reference to the explanation of ExoSAP-IT test kit, detailed step is following:
1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;
2.37 ℃ hatch 15min.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.The product that enzyme is cut after the processing directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the locus specificity primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: respectively get the corresponding ASPE primer of C19007T-w, C19007T-m, C8092A-w, C8092A-m, A2282C-w, A2282C-m, G1301A-w, G1301A-m, A1578G-w and A1578G-m stock solution 10ul respectively in the 1.5ml Eppendorf tube; Add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations.
Five, hybridization
1. according to designed ASPE primers, (microballoon concentration is 2.5 * 10 to select above-mentioned ten kinds of microballoons
5Individual/ml).Every kind of microballoon has the different colours coding respectively, and the while, every kind of microsphere surface was connected with the specific oligonucleotide sequence (anti-tag) of one section 24bp respectively, and these anti-tag sequences can combine with the tag sequence specific that corresponding ASPE primer 5 ' is held respectively;
2. the microballoon of getting every kind of numbering of 1ul respectively is in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
The 60s 7.95 ℃ unwind, 37 ℃ of hybridization 15min;
8. the microballoon after the hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul; Microballoon is in >=centrifugal the 2-5min of 3000g;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product is through Luminex serial analysis instrument detecting.With the carrier of polystyrene microsphere, as detection platform, nucleic acid molecule is carried out high-throughout many indexs parallel detection with fluorescence detector as reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the nucleic acid molecule of different things to be detected as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and detected result is shown in table 4 and table 5.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments ERCC1, ERCC2, XRCC1 and GSTP1 gene SNP originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): the sudden change ratio range is regarded as the wild-type homozygote at 0%-20%; 30%-70% is regarded as heterozygote; 80%-100% is regarded as the mutant homozygote.Compare with the liquid-phase chip result with the PCR sequencing PCR detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments gene associated with treatment effect of carboplatin mutant detected result and the sequencing result rate of coincideing originally and reaches 100%.Can detect ERCC1, ERCC2, XRCC1 and GSTP1 gene SNP type exactly it is thus clear that gene associated with treatment effect of carboplatin SNP provided by the present invention detects liquid-phase chip, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample gene type assay result
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (9)
1. a gene associated with treatment effect of carboplatin SNP detects liquid-phase chip, it is characterized in that, includes:
(1). wild-type and the special ASPE primer of mutant to the G1301A SNP site of XRCC1 gene is designed are right; Every kind of ASPE primer is made up of the specific sequence that is directed against goal gene SNP site of 3 ' end and the tag sequence of 5 ' end, and said wild-type and mutant specificity ASPE primer are to being SEQ ID NO.7 and SEQ ID NO.8;
(2). be coated with the microballoon of special anti-tag sequence respectively, said anti-tag sequence can be correspondingly with (1) in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.18;
(3). to the amplimer of the target sequence in the G1301ASNP site of XRCC1 gene.
2. gene associated with treatment effect of carboplatin SNP according to claim 1 detects liquid-phase chip, it is characterized in that primer is described in (3): to SEQ ID NO.27 and the SEQ ID NO.28 of G1301A.
3. gene associated with treatment effect of carboplatin SNP according to claim 1 detects liquid-phase chip, it is characterized in that, also is provided with the spacerarm sequence in the middle of anti-tag sequence described in (2) is connected with microballoon.
4. gene associated with treatment effect of carboplatin SNP according to claim 3 detects liquid-phase chip, it is characterized in that said spacerarm sequence is 5-10 T.
5. a gene associated with treatment effect of carboplatin SNP detects liquid-phase chip, it is characterized in that, mainly includes:
(1). the wild-type and the special ASPE primer of mutant that design respectively to the SNP site of every kind of type; Every kind of ASPE primer being made up of the specific sequence that is directed against goal gene SNP site of 3 ' end and the tag sequence of 5 ' end; Said wild-type and mutant specificity ASPE primer are to being: to SEQ ID NO.7 and the SEQ IDNO.8 of the G1301A of XRCC1 gene, and be selected to the SEQ ID NO.1 of the C19007T of ERCC1 gene and SEQ ID NO.2, to the SEQ ID NO.3 of the C8092A of ERCC1 gene and SEQ ID NO.4, to the SEQ IDNO.5 of the A2282C of ERCC2 gene and SEQ ID NO.6 with to more than a pair of among the SEQ ID NO.9 of the A1578G of GSTP1 gene and the SEQ ID NO.10;
(2). be coated with 10 kinds of microballoons of special anti-tag sequence respectively, said anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon, said anti-tag sequence can be correspondingly with (1) in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.11～SEQ ID NO.20;
(3). it is right to amplify the primer with target sequence that need to detect, corresponding SNP site: to SEQ ID NO.27 and the SEQ ID NO.28 of the G1301A of XRCC1 gene, and be selected to the SEQ ID NO.21 of the C19007T of ERCC1 gene and SEQ ID NO.22, to the SEQ ID NO.23 of the C8092A of ERCC1 gene and SEQ IDNO.24, to the SEQ ID NO.25 of ERCC2 Gene A 2282C and SEQ ID NO.26 with to more than a pair of among the SEQ ID NO.29 of the A1578G of GSTP1 gene and the SEQ ID NO.30.
6. gene associated with treatment effect of carboplatin SNP according to claim 5 detects liquid-phase chip, it is characterized in that, also is provided with the spacerarm sequence in the middle of anti-tag sequence described in (2) is connected with microballoon.
7. gene associated with treatment effect of carboplatin SNP according to claim 6 detects liquid-phase chip, it is characterized in that said spacerarm sequence is 5-10 T.
8. one kind is used for the specific sequence that gene associated with treatment effect of carboplatin SNP detects, and it is characterized in that said specific sequence includes to the SEQ ID NO.37 in the SNP site of the G1301A of XRCC1 gene and SEQ ID NO.38.
9. gene associated with treatment effect of carboplatin SNP according to claim 8 detects liquid-phase chip; It is characterized in that said specific sequence also includes: to the SEQ ID NO.31 of the C19007T of ERCC1 gene and SEQ ID NO.32, to the SEQ ID NO.33 of the C8092A of ERCC1 gene and SEQ ID NO.34, to the SEQ ID NO.35 of the A2282C of ERCC2 gene and SEQ ID NO.36 and/or to SEQ ID NO.39 and the SEQ ID NO.40 of the A1578G of GSTP1 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110301725A CN102312008A (en) | 2009-06-09 | 2009-06-09 | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110301725A CN102312008A (en) | 2009-06-09 | 2009-06-09 | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100400959A Division CN101580875B (en) | 2009-06-09 | 2009-06-09 | Specific sequence, liquid-phase chip and detection method for SNP detection of genes related to the efficacy of platinum-based drugs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102312008A true CN102312008A (en) | 2012-01-11 |
Family
ID=45425574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110301725A Pending CN102312008A (en) | 2009-06-09 | 2009-06-09 | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102312008A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080014587A1 (en) * | 2001-01-25 | 2008-01-17 | Petr Pancoska | Polynucleotides for Use as Tags and Tag Complements, Manufacture and Use Thereof |
| CN101250593A (en) * | 2008-02-03 | 2008-08-27 | 广州益善生物技术有限公司 | Papillomavirus detection and parting method as well as liquid phase chip thereof |
-
2009
- 2009-06-09 CN CN201110301725A patent/CN102312008A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080014587A1 (en) * | 2001-01-25 | 2008-01-17 | Petr Pancoska | Polynucleotides for Use as Tags and Tag Complements, Manufacture and Use Thereof |
| CN101250593A (en) * | 2008-02-03 | 2008-08-27 | 广州益善生物技术有限公司 | Papillomavirus detection and parting method as well as liquid phase chip thereof |
Non-Patent Citations (3)
| Title |
|---|
| ANA VC DUTRA, ET AL.: "Analysis of the XRCC1 gene as a modifier of the cerebral response in ischemic stroke", 《BMC MEDICAL GENETICS》 * |
| SAMANTHA L. WINSEY, ET AL.: "A Variant within the DNA Repair Gene XRCC3 Is Associated with the Development of Melanoma Skin Cancer", 《CANCER RESEARCH》 * |
| 张克，等: "晚期大肠癌患者血清XRCC 1基因多态性与奥沙利铂化疗疗效相关性的研究", 《中华肿瘤防治杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101812511B (en) | CYP3A4 gene SNP detection specific primer, liquid-phase chip and method | |
| CN101565749B (en) | CYP2C19 and ABCB1 gene SNP detection liquid-phase chip and detection method thereof | |
| CN101487052B (en) | Liquid phase chip for CYP2C9 and CYP2C19 gene mutation detection and detecting method thereof | |
| CN101781684B (en) | Liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection and detection method thereof | |
| CN101824466B (en) | Specific primer, liquid-phase chip and method for SNP detection of CYP2C9 and VKORC1 genes | |
| CN101671739B (en) | Specific sequence, liquid phase chip and method for SNP detection of TPMT gene | |
| CN101824467B (en) | CYP2D6 gene mutation detection liquid-phase chip and detection method | |
| CN101805798B (en) | Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection | |
| CN101445831B (en) | FSHR gene mutation detection liquid phase chip and detection method thereof | |
| CN103805684B (en) | The PCR primer that EML4-ALK fusion gene detects, test kit and liquid-phase chip | |
| CN101580875B (en) | Specific sequence, liquid-phase chip and detection method for SNP detection of genes related to the efficacy of platinum-based drugs | |
| CN103805688A (en) | PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene | |
| CN101812523A (en) | SNP (Single Nucleotide 0olymorphism) detection specific primer, liquid-phase chip and detection method of RYR1 (Ryanodine Receptors 1) gene | |
| CN103571923B (en) | Detection probes and detection liquid phase chip for BIM gene deletion mutation | |
| CN101812524B (en) | CYP3A5 gene SNP detection specific primer and liquid chip | |
| CN102021240B (en) | Liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 (glutathione S transferase pi) genes | |
| CN102312009B (en) | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments | |
| CN102533948A (en) | apoB (apolipoprotein B) gene SNP (Single Nucleotide Polymorphism) detection specific primer and liquid-phase chip | |
| CN102312008A (en) | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments | |
| CN102312007A (en) | Specific sequences and liquid-phase chip for SNP (single nucleotide polymorphism) detection of genes related to therapeutic effectiveness of platinum medicaments | |
| CN102021236B (en) | AGT1R (Angiotensin Type 1 Receptor) gene SNP (Single Nucleotide Polymorphism) detection liquid phase chip and specific primer | |
| CN102277413B (en) | Specific primer and liquid-phase chip for SNP detection of COX-1 genes | |
| CN102533951B (en) | BRAP and PSMA6 gene SNP detection specific primer and liquid phase chip | |
| CN103374607B (en) | ABCC (ATP (adenosine triphosphate)-binding cassette, sub-family C)1 gene mutation detection specific primers and liquid chip | |
| CN102533954B (en) | CYP17A1 (Cytochrome P450) genetic polymorphism detection specific primer and liquid-phase chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120111 |