CN101824476B - OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method - Google Patents
OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method Download PDFInfo
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Abstract
The invention discloses an OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection liquid-phase chip which mainly comprises ASPE (Allele Specific Primer Extension) primer pairs respectively designed for SNP sites of an OPRM1 gene, microballoons and an amplification primer, wherein each ASPE primer comprises a tag sequence at a 5' end and a specific primer sequence at a 3'end aiming at an SNP site of a target gene; the microballoons are respectively enveloped with a specific anti-tag sequence; and the amplification primer is used for amplifying a target sequence of the OPRM1 gene with A118G, IVS2+G31A, IVS2+G691C and/or IVS3+A615G SNP sites. The invention also provides a detection method using the liquid-phase chip and an SNP detected specific primer of the OPRM1 gene. The coincidence ratio of the detection method with a sequencing method reaches up to 100 percent, and the prepared OPRM1 gene SNP detection liquid-phase chip has excellent signal to noise ratio. Moreover, a designed probe and the anti-tag sequence basically have no cross reaction, and the parallel detection of a plurality of SNP sites can be realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete OPRM1 gene SNP detection specific primer, liquid-phase chip and the detection method of relating to.
Technical background
Mu opioid receptor (mu opioid receptor; MOR) be the main action site of opium medicine performance analgesia effect; Opioid drug is through improving the threshold of pain; Thereby reach analgesic effect, the opioid drug that is currently applied to pain caused by cancer treatment has multiple, but common have morphine, heroine, fentanyl, dolantin Dai Yin and an oxycodone etc.The gene (OPRM1) of coding mu opioid receptor is the main candidate that influences the reaction of opioid drug drug effect.Opioid drug treatment pain caused by cancer is very effective, and the pain therapy of most of patient with advanced cancer finally will carry out the transition to the 3rd ladder, promptly strong opioid drug treatment, but find in the clinical position that different patient's potent analgesics amounts are widely different.There are some researches show that the interindividual variation and the OPRM1 polymorphum of pain sensitivity and opioid drug analgesic effect, habituation etc. are closely related.
At present, the OPRM1 gene has identified more than 40 kind of SNP, wherein has 8 aminoacid sequences to change.The polymorphum of OPRM1 is relevant with methyl amphetamine dependence/psychosis and alcoholism.Klepstad etc. observe the influence of A118G sudden change to pain caused by cancer patient morphine demand; The result shows; The A118G sudden change causes MOR extracellular N-terminal the 40th amino acids to replace l-asparagine by aspartic acid; Make MOR lose a glycosylation site, carry the genotypic patient of 118G sudden change homozygote to the demand of morphine greater than 118A homozygote and the genotypic patient of heterozygote, this species diversity can not wait with time, the state of an illness, the course of disease that other factor such as morphine are treated and explain.The OPRM1 polymorphum not only also works at opiates but also in non-opium and alcohol type medicine, possibly give patient's methyl amphetamine habit-forming and alcoholism susceptibility.Simultaneously; The OPRM1 polymorphum possibly be the factor of the psychosis prediction of methyl amphetamine dependence; Wherein, IVS2+G691C sudden change relies on methyl amphetamine, psychosis/the transience psychosis shorten latent period between significant correlation, and carry the heroin addiction person that IVS2+G31A suddenlys change; Its heroine inhalation dose significantly increases than the junkies who does not carry this SNP sudden change, and the junkies who carries A118G and IVS2+G31A is more than other junkiess' medicine intake.A118G and IVS2+G31A polymorphum will directly influence the absorption behavior of opioid drug as genetic marker.In addition, also significant correlation of IVS3+A6151G and alcoholism.
Almost do not detect the launch of OPRM1 gene pleiomorphism at present both at home and abroad, most of report still is in experimental study stage, not commercialization as yet; Existing detection technique mainly is based upon on the basis of round pcr; Like direct sequencing, the shortcoming that real-time fluorescence quantitative PCR method etc., these technology exist sample be prone to pollute, false positive rate is high; Simultaneously, can not satisfy clinical needs owing to detect the limitation of flux.In addition, the detection that also can be used for the OPRM1 polymorphum based on the allelotrope discriminant analysis and the sex change HPLC of TaqMan technology, but it once can only carry out a kind of detection of sudden change, time and effort consuming.
Summary of the invention
One of the object of the invention provides the OPRM1 gene SNP detection liquid-phase chip.This liquid-phase chip can be used for detecting the variation of normal genotype and four kinds of common allele type A118G, IVS2+G31A, IVS2+G691C and IVS3+A6151G of OPRM1 gene.
The technical scheme that realizes above-mentioned purpose is following:
A kind of OPRM1 gene SNP detection liquid-phase chip mainly includes:
(A). the SNP site difference designed ASPE primers to the OPRM1 gene is right: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end; Said specific primer sequence is respectively: to the SEQ ID NO.9 in A118G site and SEQ ID NO.10, to the SEQ ID NO.11 in IVS2+G31A site and SEQID NO.12, to the SEQ ID NO.13 in IVS2+G691C site and SEQ ID NO.14 and/or to the SEQ ID NO.15 and the SEQ ID NO.16 in IVS3+A6151G site, said tag sequence is selected from the sequence among the SEQ ID NO.1-SEQ ID NO.8;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used for amplification and have: the amplimer of the OPRM1 gene target sequence in A118G, IVS2+G31A, IVS2+G691C and/or IVS3+A6151G SNP site.
Preferably, said amplimer is: to the SEQ ID NO.25 in A118G site and SEQ ID NO.26, to the SEQ ID NO.27 in IVS2+G31A site and SEQ ID NO.28, to the SEQ ID NO.29 in IVS2+G691C site and SEQ ID NO.30 and/or to the SEQ ID NO.31 and the SEQ ID NO.32 in IVS3+A6151G site.
Preferably, said ASPE primer is to being: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 and the sequence of being made up of SEQ IDNO.2 and SEQ ID NO.10, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 reach the sequence be made up of SEQ IDNO.4 and SEQ ID NO.12, be made up of SEQ ID NO.5 and SEQ ID NO.13 reaches the sequence be made up of SEQ IDNO.6 and SEQ ID NO.14 and/or reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.16 by the sequence that SEQ ID NO.7 and SEQ ID NO.15 form.
Preferably, also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
Another object of the present invention provides the method for using above-mentioned liquid-phase chip that the OPRM1 gene SNP is detected.
A kind of method of using above-mentioned liquid-phase chip to the detection of OPRM1 gene SNP mainly may further comprise the steps:
(1) pcr amplification testing sample DNA;
(2) the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing;
(3) carry out primer extension reaction with said ASPE primer, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band;
(4) microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(5) product behind the hybridization and Streptavidin-phycoerythrin react;
(6) detect through fluorescence detector.
Another object of the present invention provides the Auele Specific Primer that a kind of OPRM1 of being used for gene SNP detects.
Concrete technical scheme is following:
A kind ofly be used for the Auele Specific Primer that the OPRM1 gene SNP detects, mainly include: to the SEQ IDNO.9 in A118G site and SEQ ID NO.10, to the SEQ ID NO.11 in IVS2+G31A site and SEQ ID NO.12, to the SEQ ID NO.13 in IVS2+G691C site and SEQ ID NO.14 and/or to the SEQ IDNO.15 and the SEQ ID NO.16 in IVS3+A6151G site.
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 OPRM1 gene SNP detection liquid-phase chip has extraordinary signal-NR; And there is not cross reaction between institute's designed probe and the anti-tag sequence basically; Can realize the parallel detection in a plurality of SNP site, special coincidence detection application need.
2. designed ASPE Auele Specific Primer of the present invention has extraordinary specificity, can accurately distinguish the genotype of various types, and can under the reaction conditions of homogeneous, carry out hybridization, and does not have non-specific binding basically between the various primer, probe.
3. detection method step of the present invention is simple; One step multiplex PCR can be accomplished four amplifications with the target sequence in SNP site; Avoid many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR, thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while; Thereby the sensitivity of detection is further enhanced, and detected result more accurately and reliably.Simultaneously, the combination of multiple ASPE Auele Specific Primer makes liquid-phase chip and detection method form one and detects the intact system of effect.
Embodiment
Embodiment 1OPRM1 gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
Four kinds of common SNP sites to OPRM1: A118G, IVS2+G31A, IVS2+G691C and IVS3+A6151G, the primer sequence of designs specificity respectively.The ASPE primer is made up of " Tag+ specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1ASPE primer sequence (Tag+ specific primer sequence)
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 wild-type or mutant specific primer sequence (shown in above-mentioned table 1).All ASPE 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.
Two, the microballoon that encapsulates of anti-tag sequence
According to institute's designed ASPE specific primer sequence; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE specific primer sequence possibly form, corresponding anti-tag sequence is as shown in table 2 on eight kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
8 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.Said spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag placed the sequence of hydrophilic environments.Through the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3) are like (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T.
The process that microballoon encapsulates is following:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) 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-ethylcarbodi imide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.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 of 100ul, and [10mmol/L Tris (pH8.0), among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of target sequence with SNP site
A118G, IVS2+G31A, IVS2+G691C and the IVS3+A6151G in the common 4 kinds of SNP sites of OPRM1 gene of target detect.Wherein A118G is positioned at exons 1, and IVS2+G31A and IVS2+G691C are positioned at intron 2, and IVS3+A6151G is positioned at introne 3.Utilize Primer5.0 design four pairs of primers (seeing table 3), amplify four 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 OPRM1 gene SNP detection liquid-phase chip is to the detection of sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES(2[N-Morpholino] ethanesulfonic?acid) | Sigma?M-2933 | 0.05M | ?2.44g |
| 5M?NaOH | Fisher?SS256-500 | --- | 5 |
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 four pairs of primers of Primer5.0 design, multiplex PCR one goes on foot the target sequence that has the SNP site in exons 1, intron 2 and the introne 3 that amplifies OPRM1, and the product size is respectively 322bp, 333bp, 378bp, 395bp.Primer sequence (SEQNO.25-SEQ NO.32) 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.25-32 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
10 * damping fluid (contains Mg
2+) 5ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.5ul
Multiple PCR primer working fluid (each 16.7pmol/mL) 6ul
Template DNA (10ng/ul) 1ul
ddH
2O 33.5ul
Be total to 50ul
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 are subsequent use.
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 A118G, IVS2+G31A, IVS2+G691C and IVS3+A6151G corresponding wild type and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube; Add 10mmol/L TrisBuffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
Blended ASPE primer working fluid (each 500nmol/L) 1ul
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10.ul
Be total to 20ul
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, (microballoon concentration is 2.5 * 10 to select corresponding eight kinds of optimum microballoons
5Individual/ml);
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;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
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;
10. 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 the OPRM1 gene pleiomorphism of great amount of samples, 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 anomaly 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 OPRM1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.It is thus clear that OPRM1 gene SNP detection liquid-phase chip provided by the present invention can detect the SNP type of OPRM1 gene exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
| Sequence number NO. | A118G- w | A118G- m | IVS2+G3 1A-w | IVS2+G31 A-m | IVS2+G 691C-w | IVS2+G 691C-m | IVS3+A 6151G- w | IVS3+A 6151G- m |
| Negative to clear | 20 | 22 | 18 | 22 | 15 | 23 | 13 | 19 |
| 1 | 3238 | 231 | 2558 | 238 | 1816 | 1670 | 1227 | 1149 |
| 2 | 3281 | 233 | 2385 | 234 | 341 | 2292 | 226 | 2517 |
| 3 | 45 | 3148 | 2936 | 235 | 232 | 2327 | 2265 | 231 |
| 4 | 2762 | 258 | 3191 | 253 | 2929 | 235 | 1630 | 1536 |
| 5 | 2792 | 242 | 2970 | 262 | 1616 | 1461 | 221 | 3406 |
| 6 | 2567 | 284 | 2033 | 245 | 1562 | 1304 | 1719 | 1653 |
| 7 | 315 | 2154 | 2695 | 285 | 242 | 2084 | 241 | 2418 |
| 8 | 1284 | 1368 | 2457 | 293 | 2965 | 231 | 1540 | 1342 |
| 9 | 2565 | 314 | 42 | 2365 | 259 | 2021 | 235 | 2794 |
| 10 | 2544 | 240 | 2338 | 250 | 267 | 2377 | 1232 | 1254 |
| 11 | 3419 | 268 | 2020 | 317 | 2158 | 145 | 245 | 2646 |
| 12 | 2214 | 273 | 2219 | 242 | 453 | 2933 | 224 | 2024 |
| 13 | 3168 | 133 | 3438 | 337 | 2496 | 231 | 232 | 3397 |
| 14 | 3302 | 241 | 2193 | 36 | 230 | 2772 | 2785 | 281 |
| 15 | 3100 | 237 | 2028 | 237 | 1634 | 1788 | 139 | 2704 |
| 16 | 2886 | 307 | 3361 | 231 | 233 | 2420 | 229 | 2730 |
| 17 | 3329 | 230 | 3368 | 285 | 2856 | 235 | 268 | 2514 |
| 18 | 1542 | 1689 | 2329 | 299 | 143 | 2246 | 1556 | 1372 |
| 19 | 2881 | 244 | 2428 | 240 | 1836 | 1628 | 235 | 2338 |
| 20 | 2222 | 263 | 2151 | 256 | 142 | 2878 | 237 | 2265 |
Table 5 sample OPRM1 Polymorphism Analysis result
The liquid-phase chip of the ASPE primer that embodiment 3 is different detects the detection of transgenation to the OPRM1 gene SNP
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detection liquid-phase chip with OPRM1 Gene A 118G site mutation is an example; To the wild-type of A118G and the specific primer sequence of mutant design ASPE primer 3 ' end; The Tag sequence of ASPE primer 5 ' end then is selected from 6 among the SEQ ID NO.1-SEQID NO.8; Accordingly, encapsulate anti-tag sequence selection SEQ ID NO.17-SEQ ID NO.24 on microballoon and corresponding tag sequence complementary pairing.Specifically design shown in following table (table 6).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
The design of table 6 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, sample 21-40 detected by embodiment 2 said testing processes and method,
Detected result is following:
Table 7 pattern detection result (MFI) and Polymorphism Analysis
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 equivalence of the present invention and implement or change, all should be contained in the claim of the present invention.
Sequence table
< 110>Guangzhou Yishan Biotechnology Co., Ltd.
< 120>OPRM1 gene SNP detection specific primer, liquid-phase chip and detection method
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<210>25
<211>18
<212>DNA
< 213>artificial sequence
<400>25
ccccacgaac?gccagcaa 18
<210>26
<211>18
<212>DNA
< 213>artificial sequence
<400>26
agagccccac?cacgcaca 18
<210>27
<211>20
<212>DNA
< 213>artificial sequence
<400>27
actccccgaa?atgccaaaat 20
<210>28
<211>23
<212>DNA
< 213>artificial sequence
<400>28
cagagaaatc?agaaataaat?aga 23
<210>29
<211>21
<212>DNA
< 213>artificial sequence
<400>29
tttgtcctgc?acgaagctta?a 21
<210>30
<211>20
<212>DNA
< 213>artificial sequence
<400>30
catcagtcca?tagcacacgg 20
<210>31
<211>23
<212>DNA
< 213>artificial sequence
<400>31
ttttacaaga?catctgtgga?gag 23
<210>32
<211>23
<212>DNA
< 213>artificial sequence
<400>32
ttatgaatag?tctaaaagcc?aaa 23
Claims (6)
1. an OPRM1 gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A). the SNP site difference designed ASPE primers to the OPRM1 gene is right: every kind of ASPE primer is made up of to the specific primer sequence in goal gene SNP site the tag sequence and the 3 ' end of 5 ' end; Said specific primer sequence is respectively: to the SEQ ID NO.9 in A118G site and SEQ ID NO.10, to the SEQ ID NO.11 in IVS2+G31A site and SEQ ID NO.12, to the SEQ ID NO.13 in IVS2+G691C site and SEQ ID NO.14 and/or to the SEQ ID NO.15 and the SEQ ID NO.16 in IVS3+A6151G site, said tag sequence is selected from the sequence among the SEQ ID NO.1-SEQ ID NO.8;
(B). be coated with the microballoon of special anti-tag sequence respectively, above-mentioned every kind of microballoon has the different colours coding; Said anti-tag sequence is selected from the sequence among SEQ ID NO.17～SEQ ID NO.24, and said anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). be used to increase have A118G, the amplimer of the OPRM1 gene target sequence in IVS2+G31A, IVS2+G691C and/or IVS3+A6151G SNP site.
2. OPRM1 gene SNP detection liquid-phase chip according to claim 1; It is characterized in that said amplimer is: to the SEQ ID NO.25 in A118G site and SEQ ID NO.26, to the SEQ ID NO.27 in IVS2+G31A site and SEQ ID NO.28, to the SEQ ID NO.29 in IVS2+G691C site and SEQ ID NO.30 and/or to the SEQ ID NO.31 and the SEQ ID NO.32 in IVS3+A6151G site.
3. OPRM1 gene SNP detection liquid-phase chip according to claim 1 and 2; It is characterized in that said ASPE primer is to being: the sequence that sequence of being made up of SEQ ID NO.1 and SEQ ID NO.9 and the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.10, the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.11 reach the sequence be made up of SEQ ID NO.4 and SEQ ID NO.12, be made up of SEQ ID NO.5 and SEQ ID NO.13 reaches the sequence be made up of SEQ ID NO.6 and SEQ ID NO.14 and/or reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.16 by the sequence that SEQ ID NO.7 and SEQ ID NO.15 form.
4. OPRM1 gene SNP detection liquid-phase chip according to claim 1 is characterized in that, also is provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon.
5. OPRM1 gene SNP detection liquid-phase chip according to claim 4 is characterized in that, said spacerarm sequence is 5-10 T.
6. one kind is used for the Auele Specific Primer that the OPRM1 gene SNP detects, and includes: to the SEQ ID NO.9 in A118G site and SEQ ID NO.10, to the SEQ ID NO.11 in IVS2+G31A site and SEQ ID NO.12, to the SEQ ID NO.13 in IVS2+G691C site and SEQ ID NO.14 and/or to the SEQ ID NO.15 and the SEQ ID NO.16 in IVS3+A6151G site.
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| CN201010148416XA CN101824476B (en) | 2010-04-09 | 2010-04-09 | OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method |
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| CN201010148416XA CN101824476B (en) | 2010-04-09 | 2010-04-09 | OPRM1 (Opiate Receptor Mu 1) gene SNP (Single Nucleotide Polymorphism) detection specific primer, liquid-phase chip and detection method |
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