CN102021239A - Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection - Google Patents
Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection Download PDFInfo
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Abstract
The invention discloses a specific primmer and a liquid phase chip for solute carrier organic anion transporter 1B1 (SLCO1B1) gene signal nucleotide polymorphism (SNP) detection. The liquid phase chip mainly comprises allele specific primer extension (ASPE) primer pairs, microspheres and amplifiers, wherein each ASPE primer consists of a tag sequence at a 5' end and specific primers at a 3' end aiming at an SNP locus; the specific primers comprise SEQ ID NO.11 and SEQ ID NO.12 aiming at T521C SNP locus, SEQ ID NO.13 and SEQ ID NO.14 aiming at T89595C SNP locus, SEQ ID NO.15 and SEQ ID NO.16 aiming at A388G SNP locus, SEQ ID NO.17 and SEQ ID NO.18 aiming at C463A SNP locus, and/or SEQ ID NO.19 and SEQ ID NO.20 aiming at A1929C SNP locus; and the tag sequence is selected from SEQ ID NO.1 to SEQ ID NO.10. The coincidence rate of the liquid phase chip provided by the invention and the detection result of a sequencing method reaches 100 percent. And the prepared liquid phase chip for the SLCO1B1 gene SNP detection has high signal-to-noise ratio.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of SLCO1B1 gene test Auele Specific Primer, the liquid-phase chip of relating to.
Background technology
Solute carrier organic anion translocator family member 1B1 (Solute carrier organic anion transporter family, member 1B1, SLCO1B1), this gene coded protein organic anion transhipment polypeptide OATPIB1 (claim OATP-C again, OATP2, LST1), liver is absorbed his spit of fland regulating effect is arranged.OATP1B1 is a kind of translocator of a kind of distribution of specific on the liver cell basilar membrane, close with the liver picked-up interactively of many endogenous or exogenous material in the body, bile acide for example, sulfuration and glucuronidation binding substances, Triiodothyronine, polypeptide, Rheumatrex and statins (comprising Pravastatin, pitavastatin, rosuvastatin and simvastatin etc.) all are the substrates of OATP1B1 transhipment.In recent years, the SLCO1B1 gene is found a plurality of single nucleotide polymorphism (singlenucleotide polymorphism successively, SNP), the SNP site rs4149156 (T521C) that wherein confirms in the experiment in vitro and in vivo is relevant unusually with transport function with rs2306283 (A388G).Studies show that, have 1 C allelotrope in the rs4149156 of SLCO1B1 gene (T521C) site, compare with wild-type TT genotype patient, 4.5 times of the possibilities that patient suffers from myopathy will be improved, have 2 C allelotrope, compare with wild-type TT genotype patient, will improve 17 times of the possibilities that patient suffers from myopathy.There are some researches show that simultaneously rs11045819 (C463A) is also relevant with the possibility that myopathy takes place with rs34671512 (A 1929C).
At present the testing product of SLCO1B1 polymorphism generally is based on the PCR-restriction fragment length polymorphism of round pcr and direct sequencing etc., exist sensitivity low, the shortcoming that sample easily pollutes, false positive rate is high owing to detect the limitation of flux, can not satisfy the needs of practical application simultaneously.
Summary of the invention
One of purpose of the present invention provides SLCO1B1 and detects liquid-phase chip because of SNP.This liquid-phase chip can be used for detecting wild-type and the mutant of SLCO1B1 gene five kinds of common genotype T521C, T89595C, A388G, C463A and A1929C.
Realize that the above-mentioned purpose technical scheme is as follows:
A kind of SLCO1B1 gene SNP detection liquid-phase chip mainly includes:
(A) at the SNP site of SLCO1B1 gene, the ASPE primer of She Ji wild-type and mutant is right respectively: every kind of ASPE primer is made up of at the Auele Specific Primer in SNP site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.11 and the SEQ ID NO.12 in T521C SNP site, SEQ ID NO.13 and SEQ ID NO.14 at T89595C SNP site, SEQ ID NO.15 and SEQ ID NO.16 at A388G SNP site, SEQ ID NO.17 and SEQ ID NO.18 at C463A SNP site, and/or at the SEQ ID NO.19 and the SEQ IDNO.20 in A1929C SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.10;
(B) be coated with microballoon special anti-tag sequence, that have the different colours coding respectively, described anti-tag sequence is selected from the sequence among SEQID NO.21～SEQ ID NO.30, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C) be used to amplify the amplimer of the SLCO1B1 gene target sequence in SNP site with T521C, T89595C, A388G, C463A and/or A1929C.
Preferably, described amplimer is: at the SEQ ID NO.31 in T521C SNP site and SEQ ID NO.32, at the SEQ ID NO.33 in T89595C SNP site and SEQ ID NO.34, at the SEQ IDNO.35 in A388G SNP site and SEQ ID NO.36, at the SEQ ID NO.37 in C463A SNP site and SEQ ID NO.38 and/or at the SEQ ID NO.39 and the SEQ ID NO.40 in A1929C SNP site.
Preferably, described ASPE primer is to being: at the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.11 in T521C SNP site and the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.12, at the sequence of forming by SEQID NO.3 and SEQ ID NO.13 in T89595C SNP site and the sequence of forming by SEQ ID NO.4 and SEQ ID NO.14, at the sequence of forming by SEQ ID NO.5 and SEQ ID NO.15 in A388G SNP site and the sequence of forming by SEQ ID NO.6 and SEQ IDNO.16, at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.17 in C463A SNP site and the sequence of forming by SEQ ID NO.8 and SEQ ID NO.18, and/or at the sequence of forming by SEQ ID NO.9 and SEQ ID NO.19 in A1929C SNP site and the sequence of forming by SEQ ID NO.10 and SEQ ID NO.20.
Another object of the present invention provides and is used for the Auele Specific Primer that the SLCO1B1 gene SNP detects.
Concrete technical scheme is as follows:
Be used for the Auele Specific Primer that the SLCO1B1 gene SNP detects, mainly include: at the SEQ ID NO.11 in T521C SNP site and SEQ ID NO.12, at the SEQ ID NO.13 in T89595C SNP site and SEQ ID NO.14, at the SEQ ID NO.15 in A388G SNP site and SEQ ID NO.16, at the SEQ ID NO.17 in C463A SNP site and SEQ ID NO.18 and/or at the SEQ ID NO.19 and the SEQ ID NO.20 in A1929C SNP site.
Major advantage of the present invention is:
1. the detected result of detection liquid-phase chip provided by the present invention and sequencing is coincide rate up to 100%.Prepared SLCO1B1 gene SNP detection liquid-phase chip has extraordinary signal-noise ratio, and there is not cross reaction between designed probe and the anti-tag sequence basically, choosing of tag sequence label, anti-tag sequence label and combining of tag sequence label and concrete ASPE primer, can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the ASPE type specificity primer of the present invention's design has extraordinary specificity, can accurately distinguish the genotype of various types.
3. the detection method step of using liquid-phase chip of the present invention is simple, five kinds of SNPs detect and can finish five amplifications that contain the target sequence in SNP site by a step multiplex PCR, 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 feature of accurate while.
Liquid-phase chip of the present invention the needed time of detection method well below sequencing technologies commonly used, realistic especially application need.
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 different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 SLCO1B1 gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
Wild-type and mutant at five kinds of common genotype T521C (rs4149056), T89595C (rs4363657), A388G (rs2306283), C463A (rs11045819) and the A1929C (rs34671512) of SLCO1B1 gene design specific primer sequence respectively.The ASPE primer is made up of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence of table 1 SLCO1B1 (Tag sequence+specific primer sequence)
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence at anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (shown in above-mentioned table 1) of wild-type.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/LTris Buffer.
Two, the microballoon of anti-tag sequence bag quilt
According to 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 may form, corresponding anti-tag sequence is as shown in table 2 on 10 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
Ten kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag sequence bag by on microballoon.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly add the spacerarm sequence of the preceding paragraph 5-10 T before each anti-tag sequence, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag is placed the sequence of hydrophilic environments.By 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 as (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, and the process of microballoon bag quilt is as follows:
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-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, 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 [10mmol/L Tris (pH8.0)] of 100ul, and among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
SNP mutational site at five kinds of common genotype T521C (rs4149056), T89595C (rs4363657), A388G (rs2306283), C463A (rs11045819) and the A1929C (rs34671512) of SLCO1B1 gene, utilize the Primer5.0 design of amplification primers to (seeing Table 3), amplify five target sequences that contain the SNP site respectively.
Table 3 amplifies the primer that has the target sequence in SNP site in the SLCO1B1 gene
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/LTrisBuffer.
Embodiment 2 utilization SLCO1B1 gene test liquid-phase chips are to the detection of sample
The prescription of described various solution is as follows:
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 |
| 5MNaCl | 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:
With reference to " molecular cloning " methods involving, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Utilize five pairs of primers of Primer5.0 design, multiplex PCR one step amplifies five kinds of common SNP mutational site T521C, T89595C, A388G, C463A and A1929C totally five the target sequence that contains the SLCO1B1 gene respectively, the product size is respectively 443bp, 395bp, 445bp, 414bp and 246bp, and primer sequence (SEQ ID NO.31-40) 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 ID NO.31-40 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is as follows:
2 * damping fluid (contains Mg
2+) 25ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.2ul
Multiple PCR primer working fluid (each 8.3pmol/mL) 6ul
Template DNA (10ng/ul) 2ul
ddH
2O 12.g?ul
Be total to 50ul
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are standby.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
2.37 ℃ hatch 15min, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE 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: get five kinds of common genotype T521C, T89595C, A388G, C463A and the A1929C SNP site corresponding wild type of SLCO1B1 gene to be detected and mutant ASPE primer 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 as follows:
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 10ul
Be total to 20ul
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are standby.
Five, hybridization
1. according to the ASPE primer of design, (microballoon concentration is 2.5 * 10 to the corresponding optimum microballoon of every group selection
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 25ui
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. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, 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 by Luminex serial analysis instrument detecting.Detected result is shown in table 4, table 5 and table 6.
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 determined threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments SLCO1B1 gene SNP site originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with of threshold value (cut-off value) is as follows: the sudden change ratio range is considered as the wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as the anomaly homozygote.Compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments SLCO1B1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen SLCO1B1 gene SNP detection liquid-phase chip provided by the present invention can detect the SNP type of SLCO1B1 exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample SLCO1B1 transgenation ratio (%)
Table 6 sample SLCO1B1 gene mutation type analytical results
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of SLCO1B1 gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with SLCO1B1 gene T521C site mutation is example, respectively at the wild-type of T521C and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is selected from SEQ ID NO.1-SEQ IDNO.10, accordingly, bag is selected from SEQ ID NO.21-SEQID NO.30 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Specific design is shown in following table (table 7).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
The design of table 7 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 21-40 is detected, detected result is as follows:
Table 8 pattern detection result and Polymorphism Analysis
Other is at the liquid-phase chip in different SNP sites, and the ASPE primer uses different Tag sequences, and its result is still reliable and stable, and concrete data are omitted.
Embodiment 4 different interval arm liquid-phase chips detect SLCO1B1 gene SNP site SNP
One, the design (selection of spacerarm) of liquid-phase chip preparation
Detection liquid-phase chip with SLCO1B1 gene T388G site mutation is an example, verifies the influence that different spacerarm liquid-phase chips detects the SLCO1B1 gene SNP.At the wild-type of T388G and the specific primer sequence of mutant design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end then is respectively SEQ ID NO.5 and SEQ ID NO.6, accordingly, bag is respectively SEQ ID NO.25 and SEQ ID NO.26 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Verify the influence that different spacerarm liquid-phase chips detects the SLCO1B1 gene SNP, wherein, different spacerarms is (CH2) 12 or 5-10 T, and specific design is shown in following table (table 9).Synthetic, the anti-tag sequence bag of ASPE primer is described like embodiment 1 and embodiment 2 by microballoon, amplimer, detection method.
The design of table 9 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned design preparation, by embodiment 2 described testing processes and method sample 41-60 is detected, detected result is as follows:
Table 10 pattern detection result and Polymorphism Analysis
Spacerarm is the liquid-phase chip of (CH2) 12 among the embodiment 4, its detected result is reliable and stable, and (other spacerarm liquid-phase chip detected result is also like this, concrete data are omitted), and the hybridization rate of the spacerarm of preferred 5-10 T and hybridization specificity all are better than the liquid-phase chip of spacerarm for (CH2) 12, and the spacerarm of a 5-10 of the present invention T all is better than other spacerarms.
More than be at 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.
Sequence table
＜110〉Guangzhou Yishan Biotechnology Co., Ltd.
＜120〉SLC01B1 gene SNP detection specific primer and liquid-phase chip
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＜213〉artificial sequence
<400>37
ttcaacatcg?accttatcca?c 21
<210>38
<211>19
<212>DNA
＜213〉artificial sequence
<400>38
gggctgcctg?tgtgttctc 19
<210>39
<211>20
<212>DNA
＜213〉artificial sequence
<400>39
gggtctactt?gggcttgtct 20
<210>40
<211>20
<212>DNA
＜213〉artificial sequence
<400>40
tggaaacaca?gaagcagaag 20
Claims (5)
1. a SLCO1B1 gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A) at the SNP site of SLCO1B1 gene, the ASPE primer of She Ji wild-type and mutant is right respectively: every kind of ASPE primer is made up of at the Auele Specific Primer in SNP site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.11 and the SEQ ID NO.12 in T521C SNP site, SEQ IDNO.13 and SEQ ID NO.14 at T89595C SNP site, SEQ ID NO.15 and SEQ ID NO.16 at A388G SNP site, SEQ ID NO.17 and SEQ ID NO.18 at the C463ASNP site, and/or at the SEQ ID NO.19 and the SEQ ID NO.20 in A1929C SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.10;
(B) be coated with microballoon special anti-tag sequence, that have the different colours coding respectively, described anti-tag sequence is selected from the sequence among SEQID NO.21～SEQ ID NO.30, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C) be used to amplify the amplimer of the SLCO1B1 gene target sequence in SNP site with T521C, T89595C, A388G, C463A and/or A1929C.
2. SLCO1B1 gene SNP detection liquid-phase chip according to claim 1, it is characterized in that described amplimer is: at the SEQ ID NO.31 in T521C SNP site and SEQ ID NO.32, at the SEQ ID NO.33 in T89595C SNP site and SEQ ID NO.34, at the SEQ ID NO.35 in A388G SNP site and SEQ ID NO.36, at the SEQ ID NO.37 in C463A SNP site and SEQ ID NO.38 and/or at the SEQ ID NO.39 and the SEQ IDNO.40 in A1929C SNP site.
3. SLCO1B1 gene SNP detection liquid-phase chip according to claim 1, it is characterized in that described ASPE primer is to being: at the sequence of forming by SEQ IDNO.1 and SEQ IDNO.11 in T521C SNP site and the sequence of forming by SEQ IDNO.2 and SEQID NO.12, at the sequence of forming by SEQ ID NO.3 and SEQ ID NO.13 in T89595C SNP site and the sequence of forming by SEQ ID NO.4 and SEQ ID NO.14, at the sequence of forming by SEQ ID NO.5 and SEQID NO.15 in A388G SNP site and the sequence of forming by SEQ ID NO.6 and SEQ ID NO.16, at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.17 in C463A SNP site and the sequence of forming by SEQ ID NO.8 and SEQ ID NO.18, and/or at the sequence of forming by SEQ IDNO.9 and SEQ ID NO.19 in A1929C SNP site and the sequence of forming by SEQ IDNO.10 and SEQ ID NO.20.
4. according to each described SLCO1B1 gene SNP detection liquid-phase chip of claim 1-3, it is characterized in that described spacerarm is 5-10 T.
5. one kind is used for the Auele Specific Primer that the SLCO1B1 gene SNP detects, it is characterized in that, mainly include: at the SEQ ID NO.11 in T521C SNP site and SEQ ID NO.12, at the SEQ ID NO.13 in T89595C SNP site and SEQ IDNO.14, at the SEQ ID NO.15 in A388G SNP site and SEQ ID NO.16, at the SEQ IDNO.17 in C463A SNP site and SEQ ID NO.18 and/or at the SEQ ID NO.19 and the SEQ ID NO.20 in A1929C SNP site.
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| CN201010196840.1A CN102021239B (en) | 2010-06-08 | 2010-06-08 | Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection |
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| CN201010196840.1A CN102021239B (en) | 2010-06-08 | 2010-06-08 | Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection |
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| CN102021239B CN102021239B (en) | 2014-01-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146482A (en) * | 2011-04-29 | 2011-08-10 | 广州益善生物技术有限公司 | IL-23R gene polymorphism detection specific primer and liquid phase chip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070244310A1 (en) * | 2001-01-25 | 2007-10-18 | Petr Pancoska | Polynucleotides for use tags and tag complements, manufacture and use thereof |
| CN101619352A (en) * | 2009-01-23 | 2010-01-06 | 中南大学 | Double-probe gene mutation detecting method based on allele special amplification as well as special chip and kit thereof |
| CN101717816A (en) * | 2009-03-09 | 2010-06-02 | 中南大学 | Gene detection chip of OATP1B1 major gene mutation |
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2010
- 2010-06-08 CN CN201010196840.1A patent/CN102021239B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070244310A1 (en) * | 2001-01-25 | 2007-10-18 | Petr Pancoska | Polynucleotides for use tags and tag complements, manufacture and use thereof |
| CN101619352A (en) * | 2009-01-23 | 2010-01-06 | 中南大学 | Double-probe gene mutation detecting method based on allele special amplification as well as special chip and kit thereof |
| CN101717816A (en) * | 2009-03-09 | 2010-06-02 | 中南大学 | Gene detection chip of OATP1B1 major gene mutation |
Non-Patent Citations (1)
| Title |
|---|
| 《Pharmacogenomics》 20051231 Robert AM Op den Bujjsch et al. Rapid genotyping of the OATP1B1 polymorphisms A388G and T521C with real-time PCR FRET assays 393-397 第6卷, 第4期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146482A (en) * | 2011-04-29 | 2011-08-10 | 广州益善生物技术有限公司 | IL-23R gene polymorphism detection specific primer and liquid phase chip |
| CN102146482B (en) * | 2011-04-29 | 2013-02-27 | 广州益善生物技术有限公司 | IL-23R gene polymorphism detection specific primer and liquid phase chip |
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| CN102021239B (en) | 2014-01-01 |
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