CN102021237A - Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes - Google Patents
Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes Download PDFInfo
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Abstract
The invention discloses a specificity primer and a liquid phase chip for SNP detection of CYP4F2 gene. The liquid phase chip comprises an ASPE primer formed by the specificity primer aiming at a target gene SNP locus of the tag sequence on the 5' end and 3' end, wherein the specificity primer is SEQ ID NO.13 and SEQ ID NO.14 aiming at the G1297A SNP locus and/or SEQ ID NO.15 and SEQ ID NO.16 aiming at the T34G SNP locus, a microsphere and an amplification primer. The invention also provides a liquid phase chip for SNP detection of CYP4F2 and EPHX1 genes, which comprises the corresponding compositions of the liquid phase chip for the SNP detection of the CYP4F2 and EPHX1 genes, an ASPE primer pair aiming at the G357A, G19512990A, T337C and/or A416G SNP locus of the EPHX1 gene, microsphere and amplification primer. The detection liquid phase chip has an excellent signal-noise ratio, can avoid cross reaction and can realize the parallel detection of a plurality of SNP loca.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of CYP4F2 and EPHX1 gene test liquid-phase chip and the Auele Specific Primer of relating to.
Background technology
Cytochrome P450 4F2 (Cytochrome P450, CYP4F2) be one of environment metabolic enzyme Cytochrome P450 superfamily member, be human kidney's metabolism arachidonic acid (arachidonic acid, AA) produce 20-hydroxyl eicosatetraenoic acid (20-hydroxyeicosatetraenoic acid, 20-HETE) topmost enzyme, 20-HETE has vasoconstriction and regulates the vital role of ion stable state, kidney 20-HETE synthetic is not enough and hypertensive being related closely, so CYP4F2 becomes the important factor in order that hypertension takes place.Studies show that the W12G and the V433M sudden change that occur in CYP4F2 will cause the 20-HETE comparison according to the amount that is reduced to 56%-66%.Wherein, the W12G of CYP4F2 (rs3093105 T34G) is CYP4F2*2, and V433M (rs2108622 G1297A) occurs in exon 2.
(Microsomal epoxide hydrolase, mEH are a kind of II phase metabolic enzymes of important bio-transformation EPHX1) to microsome epoxide hydrolase, and this enzyme has high conservative by the EPHX1 genes encoding that is positioned human chromosomal 1q42.1.The multiple epoxidation intermediate product of its catalysis is hydrolyzed to trans dihydrodiol more soluble in water and excretes.Studies show that the polymorphism of EPHX1 gene coding region is by influencing the activity that proteinic stability changes enzyme.The T of exon 3 → C transformation makes Tyr113, and (rs1051740 T337C), causes enzymic activity to descend 39% by the His replacement; The A of exon 4 → G changes makes His139 be replaced by Arg that (rs2234922 A416G), causes enzymic activity to raise 25%, there are some researches show that the decline that the EPHX1 enzyme is lived helps preventing lung cancer, but this polymorphism is also uncorrelated with the risk of sporadic colorectal cancer.
At present the testing product of CYP4F2 and EPHX1 polymorphism generally is based on fluorescence quantitative PCR method, RFLP method and the dna sequencing method etc. of round pcr, 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 a kind of CYP4F2 gene SNP detection liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of two kinds of common genotype G1297A of CYP4F2 gene and T34G.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of CYP4F2 gene SNP detection liquid-phase chip mainly includes:
(A) at the SNP site of CYP4F2 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 goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.13 in G1297A SNP site and SEQ ID NO.14 and/or at the SEQID NO.15 and the SEQ ID NO.16 in T34G SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQID NO.12;
(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.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to amplify the amplimer of the CYP4F2 gene target sequence in SNP site with G1297A and/or T34G.
Preferably, described amplimer is the SEQ ID NO.37～SEQ ID NO.38 at G1297A SNP site, and/or at T34G SNP site SEQ ID NO.39～SEQ ID NO.40.
More preferably, described ASPE primer is to being: the sequence of forming at the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.13 in G1297A SNP site and by SEQ ID NO.2 and SEQ ID NO.14 and/or at the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.15 in T34G SNP site and the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.16.
Another object of the present invention provides a kind of CYP4F2 and EPHX1 gene SNP detection liquid-phase chip, and this liquid-phase chip can carry out synchronous detection to CYP4F2 and EPHX1 gene.
Concrete technical scheme is as follows:
A kind of CYP4F2 and EPHX1 gene SNP detection liquid-phase chip mainly include:
(A) at the SNP site of CYP4F2 gene and EPHX1 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 goal gene mutational site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.13 in CYP4F2 gene G1297A SNP site and SEQ ID NO.14 and/or at the SEQ ID NO.15 and the SEQ ID NO.16 in CYP4F2 gene T34G SNP site; And at the SEQ ID NO.17 in EPHX1 gene G357A SNP site and SEQ ID NO.18, at the SEQ ID NO.19 in EPHX1 gene G19512990A SNP site and SEQ ID NO.20, at the SEQ ID NO.21 in EPHX1 gene T337C SNP site and SEQ ID NO.22 and/or at the SEQ IDNO.23 and the SEQ ID NO.24 in EPHX1 gene A 416G SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.12;
(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.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to the increase amplimer of CYP4F2 gene target sequence in SNP site with G1297A and/or T34G; And the amplimer of the EPHX1 gene target sequence in the SNP site of being used to increase with G357A, G19512990A, T337C and/or A416G.
Preferably, described amplimer is at the SEQ ID NO.37～SEQ IDNO.38 in CYP4F2 gene G1297A SNP site and/or at CYP4F2 gene T34G SNP site SEQ ID NO.39～SEQ ID NO.40; And at the SEQ ID NO.41 in EPHX1 gene G357ASNP site and SEQ ID NO.42, at the SEQ ID NO.43 in EPHX1 gene G19512990ASNP site and SEQ ID NO.44, at the SEQ ID NO.45 in EPHX1 gene T337C SNP site and SEQ ID NO.46 and/or at the SEQ ID NO.47 and the SEQ ID NO.48 in EPHX1 gene A 416G SNP site.
Further, described ASPE primer is to being: the sequence of forming at the sequence of being made up of SEQ ID NO.1 and SEQ IDNO.13 in G1297A SNP site and by SEQ ID NO.2 and SEQ ID NO.14 and/or at the sequence of being made up of SEQ ID NO.3 and SEQ ID NO.15 in T34G SNP site and the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.16; And at the sequence of forming by SEQ ID NO.5 and SEQ ID NO.17 in EPHX1 gene G357ASNP site and the sequence of forming by SEQ ID NO.6 and SEQ ID NO.18, at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.19 in EPHX1 gene G19512990A SNP site and the sequence of forming by SEQ ID NO.8 and SEQ ID NO.20, at the sequence of forming by SEQ ID NO.9 and SEQ ID NO.21 in EPHX1 gene T337C SNP site and the sequence of forming by SEQ IDNO.10 and SEQ ID NO.22, and/or at the sequence of forming by SEQ ID NO.11 and SEQ ID NO.23 in EPHX1 gene A 416G SNP site and the sequence of forming by SEQ ID NO.12 and SEQ ID NO.24.
Preferably, described spacerarm sequence is 5-10 T.
Another object of the present invention provides and is used for the Auele Specific Primer that the CYP4F2 gene SNP detects.
Concrete technical scheme is as follows:
Be used for the Auele Specific Primer that the CYP4F2 gene SNP detects, include at the SEQ ID NO.13 in G1297A SNP site and SEQ ID NO.14 and/or at the SEQ ID NO.15 and the SEQ ID NO.16 in T34G SNP site.
Major advantage of the present invention is:
1. the identical rate of detection liquid-phase chip provided by the present invention and sequencing is up to 100%.Prepared CYP4F2 gene SNP detection liquid-phase chip and CYP4F2 and EPHX1 gene SNP detection liquid-phase chip have 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. detection method step of the present invention is simple, in CYP4F2 and EPHX1 gene SNP detection liquid-phase chip, six kinds of SNPs detect and can finish six 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.
4. the needed time of detection method provided by the present invention is well below sequencing technologies commonly used, realistic especially application need; And being used in combination of multiple ASPE Auele Specific Primer makes liquid-phase chip and detection method form an intact system of detection effect.
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 1CYP4F2 and EPHX1 gene SNP detection liquid-phase chip mainly include:
One, ASPE primer
SNP site G357A (rs2292566), G19512990A (rs4653436), T337C (rs1051740) and A416G (rs2234922) at two kinds of common SNP site G1297A (rs2108622) of CYP4F2 gene and T34G (rs3093105), EPHX1 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:
Table 1 ASPE primer sequence (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/L Tris 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 12 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
12 kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag sequence bag by with microballoon on.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
Two kinds of common SNP site G1297A (rs2108622) and T34G (rs3093105) at the CYP4F2 gene, four kinds of common SNP site G357A (rs2292566) of EPHX1 gene, G19512990A (rs4653436), T337C (rs1051740) and A416G (rs2234922), utilize the Primer5.0 design of amplification primers to (seeing Table 3), amplify six target sequences that contain the SNP site respectively.
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/LTrisBuffer.
In like manner, those skilled in the art can under the situation that does not comprise the EPHX1 Gene Partial, make up CYP4F2 gene test liquid-phase chip according to the method described above.
Embodiment 2 utilization embodiment 1 described CYP4F2 and EPHX1 gene test liquid-phase chip are as follows to the prescription of the described various solution of detection of sample:
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 |
| 5MNaOH | 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 six pairs of primers of Primer5.0 design, multiplex PCR one step amplifies four kinds of SNP sites (G357A, G19512990A, T337C and A416G) totally six the target sequence that contains two kinds of SNP sites of CYP4F2 gene (G1297A and T34G), EPHX1 gene respectively, the product size is respectively 345bp, 255bp, 450bp, 312bp, 236bp and 287bp, and primer sequence (SEQ ID NO.37-48) 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.37-48 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 12.8ul
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 the SNP site corresponding wild type of CYP4F2 gene to be detected and EPHX1 gene and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube, add 10mmol/LTris 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, 58 ℃ 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).Every kind of microballoon has the different colours coding respectively, and every kind of microsphere surface is connected with the specific oligonucleotide sequence (anti-tag) of one section 24bp respectively simultaneously, and these anti-tag sequences can be respectively and the tag sequence specific combination of corresponding ASPE primer 5 ' end;
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. 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 CYP4F2 and EPHX1 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 this CYP4F2 and the identical rate of EPHX1 genotype detection result and sequencing result of 20 increments and reaches 100%.As seen CYP4F2 provided by the present invention and EPHX1 gene SNP detection liquid-phase chip can detect the SNP type of CYP4F2 and EPHX1 exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample CYP4F2 and EPHX1 transgenation ratio (%)
Table 6 sample CYP4F2 and EPHX1 gene mutation type analytical results
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of CYP4F2 and EPHX1 gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with CYP4F2 gene G1297A site and EPHX1 gene G357A site mutation is example, respectively at the wild-type in G1297A SNP site and G357A SNP site 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 ID NO.12, accordingly, bag is selected from SEQ ID NO.25-SEQ ID NO.36 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
Table 9 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 the CYP4F2 gene SNP
One, the design (selection of spacerarm) of liquid-phase chip preparation
Detection liquid-phase chip with CYP4F2 gene T34G site mutation is an example, verifies the influence that different spacerarm liquid-phase chips detects the CYP4F2 gene SNP.At the wild-type of T34G 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.3 and SEQ ID NO.4, accordingly, bag is respectively SEQ ID NO.27 and SEQ ID NO.28 by the anti-tag sequence with corresponding tag sequence complementary pairing on microballoon.Verify the influence that different spacerarm liquid-phase chips detects the CYP4F2 gene SNP, wherein, different spacerarms is (CH2) 12 or 5-10 T, and specific design is shown in following table (table 10).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 10 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 11 pattern detection result and Polymorphism Analysis
The spacerarm of embodiment 4 is the liquid-phase chip of (CH2) 12, its detected result is reliable and stable, and (detected result of other spacerarm 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〉CYP4F2 and EPHX1 gene SNP detection liquid-phase chip and Auele Specific Primer
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＜213〉artificial sequence
<400>17
accctcactt?caagactaag 20
<210>18
<211>20
<212>DNA
＜213〉artificial sequence
<400>18
accctcactt?caagactaaa 20
<210>19
<211>24
<212>DNA
＜213〉artificial sequence
<400>19
taatttattc?taaagagcac?acta 24
<210>20
<211>24
<212>DNA
＜213〉artificial sequence
<400>20
taatttattc?taaagagcac?actg 24
<210>21
<211>20
<212>DNA
＜213〉artificial sequence
<400>21
ggtggagatt?ctcaacagat 20
<210>22
<211>20
<212>DNA
＜213〉artificial sequence
<400>22
ggtggagatt?ctcaacagac 20
<210>23
<211>17
<212>DNA
＜213〉artificial sequence
<400>23
cagctgcccg?caggcca 17
<210>24
<211>17
<212>DNA
＜213〉artificial sequence
<400>24
cagctgcccg?caggccg 17
<210>25
<211>24
<212>DNA
＜213〉artificial sequence
<400>25
aaagaaagga?tttgtagtaa?gatt 24
<210>26
<211>24
<212>DNA
＜213〉artificial sequence
<400>26
aaaggattaa?agtgaagtaa?ttga 24
<210>27
<211>24
<212>DNA
＜213〉artificial sequence
<400>27
aaagttgaga?tttgaatgat?tgaa 24
<210>28
<211>24
<212>DNA
＜213〉artificial sequence
<400>28
tgaagatttg?aagtaattga?aaag 24
<210>29
<211>24
<212>DNA
＜213〉artificial sequence
<400>29
atgagattat?tggatttgta?gatt 24
<210>30
<211>24
<212>DNA
＜213〉artificial sequence
<400>30
gtatttgagt?aagtaattga?ttga 24
<210>31
<211>24
<212>DNA
＜213〉artificial sequence
<400>31
attgattgtg?aatgaaatga?attg 24
<210>32
<211>24
<212>DNA
＜213〉artificial sequence
<400>32
attgttgaaa?agtgtaatga?ttga 24
<210>33
<211>24
<212>DNA
＜213〉artificial sequence
<400>33
aaaggtaaga?ttattgatga?aaag 24
<210>34
<211>24
<212>DNA
＜213〉artificial sequence
<400>34
attgatgagt?atattgtgta?gtaa 24
<210>35
<211>24
<212>DNA
＜213〉artificial sequence
<400>35
attgtgaagt?ataaagatga?ttga 24
<210>36
<211>24
<212>DNA
＜213〉artificial sequence
<400>36
tgatgatttg?aatgaagatt?gatt 24
<210>37
<211>20
<212>DNA
＜213〉artificial sequence
<400>37
ccttggagag?acagacagtt 20
<210>38
<211>18
<212>DNA
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<400>38
gtcatctccc?gccatgtc 18
<210>39
<211>20
<212>DNA
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<400>39
ctgcctctcc?actgtccctg 20
<210>40
<211>19
<212>DNA
＜213〉artificial sequence
<400>40
ttccgtcttg?ggggttgtg 19
<210>41
<211>20
<212>DNA
＜213〉artificial sequence
<400>41
cgataagttc?cgtttcaccc 20
<210>42
<211>20
<212>DNA
＜213〉artificial sequence
<400>42
tcatctccat?ccttcctctc 20
<210>43
<211>20
<212>DNA
＜213〉artificial sequence
<400>43
aagattgcct?cccacacatt 20
<210>44
<211>20
<212>DNA
＜213〉artificial sequence
<400>44
taatcatcca?tccagcgttg 20
<210>45
<211>20
<212>DNA
＜213〉artificial sequence
<400>45
gctgcttcca?ctatggcttc 20
<210>46
<211>20
<212>DNA
＜213〉artificial sequence
<400>46
attgggttct?gaatctctcc 20
<210>47
<211>21
<212>DNA
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<400>47
actaagggtg?gcaggactca?a 21
<210>48
<211>20
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ggcagatgac?ttcaaaaacg 20
Claims (9)
1. a CYP4F2 gene SNP detection liquid-phase chip is characterized in that, mainly includes:
(A) at the SNP site of CYP4F2 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 goal gene SNP site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.13 in G1297A SNP site and SEQ ID NO.14 and/or at the SEQID NO.15 and the SEQ ID NO.16 in T34G SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.12;
(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.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to amplify the amplimer of the CYP4F2 gene target sequence in SNP site with G1297A and/or T34G.
2. CYP4F2 gene SNP detection liquid-phase chip according to claim 1, it is characterized in that, described amplimer is the SEQ ID NO.37～SEQ ID NO.38 at G1297A SNP site, and/or at T34G SNP site SEQ ID NO.39～SEQ ID NO.40.
3. CYP4F2 gene SNP detection liquid-phase chip according to claim 1, it is characterized in that described ASPE primer is to being: the sequence of forming at the sequence of forming by SEQ ID NO.1 and SEQ ID NO.13 in G1297A SNP site and by SEQ ID NO.2 and SEQ ID NO.14 and/or at the sequence of forming by SEQ ID NO.3 and SEQ ID NO.15 in T34G SNP site and the sequence of forming by SEQ ID NO.4 and SEQ ID NO.16.
4. according to each described CYP4F2 gene SNP detection liquid-phase chip of claim 1-3, it is characterized in that described spacerarm sequence is 5-10 T.
5. CYP4F2 and EPHX1 gene SNP detection liquid-phase chip is characterized in that, mainly include:
(A) at the SNP site of CYP4F2 gene and EPHX1 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 goal gene mutational site the tag sequence and the 3 ' end of 5 ' end, and described Auele Specific Primer is: at the SEQ ID NO.13 in CYP4F2 gene G1297A SNP site and SEQ ID NO.14 and/or at the SEQ ID NO.15 and the SEQ ID NO.16 in CYP4F2 gene T34G SNP site; And at the SEQ ID NO.17 in EPHX1 gene G357ASNP site and SEQ ID NO.18, at the SEQ ID NO.19 in EPHX1 gene G19512990A SNP site and SEQ ID NO.20, at the SEQ ID NO.21 in EPHX1 gene T337C SNP site and SEQ ID NO.22 and/or at the SEQ IDNO.23 and the SEQ ID NO.24 in EPHX1 gene A 416G SNP site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.12;
(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.25～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing, and described anti-tag sequence also is provided with the spacerarm sequence in the middle of being connected with microballoon;
(C) be used to the increase amplimer of CYP4F2 gene target sequence in SNP site with G1297A and/or T34G; And the amplimer of the EPHX1 gene target sequence in the SNP site of being used to increase with G357A, G19512990A, T337C and/or A416G.
6. CYP4F2 according to claim 5 and EPHX1 gene SNP detection liquid-phase chip, it is characterized in that described amplimer is at the SEQ ID NO.37～SEQ ID NO.38 in CYP4F2 gene G1297A SNP site and/or at CYP4F2 gene T34G SNP site SEQ ID NO.39～SEQ ID NO.40; And at the SEQ ID NO.41 in EPHX1 gene G357ASNP site and SEQ ID NO.42, at the SEQ IDNO.43 in EPHX1 gene G19512990A SNP site and SEQ ID NO.44, at the SEQ ID NO.45 in EPHX1 gene T337C SNP site and SEQ ID NO.46 and/or at the SEQ ID NO.47 and the SEQ ID NO.48 in EPHX1 gene A 416G SNP site.
7. CYP4F2 according to claim 5 and EPHX1 gene SNP detection liquid-phase chip, it is characterized in that described ASPE primer is to being: the sequence of forming at the sequence of forming by SEQ ID NO.1 and SEQ ID NO.13 in G1297A SNP site and by SEQ ID NO.2 and SEQ ID NO.14 and/or at the sequence of forming by SEQ ID NO.3 and SEQ ID NO.15 in T34G SNP site and the sequence of forming by SEQ ID NO.4 and SEQ ID NO.16; And at the sequence of forming by SEQ ID NO.5 and SEQ ID NO.17 in EPHX1 gene G357A SNP site and the sequence of forming by SEQ IDNO.6 and SEQ ID NO.18, at the sequence of forming by SEQ ID NO.7 and SEQ ID NO.19 in EPHX1 gene G19512990A SNP site and the sequence of forming by SEQ ID NO.8 and SEQ ID NO.20, at the sequence of forming by SEQ ID NO.9 and SEQ ID NO.21 in EPHX1 gene T337C SNP site and the sequence of forming by SEQ ID NO.10 and SEQID NO.22, and/or at the sequence of forming by SEQ ID NO.11 and SEQ IDNO.23 in EPHX1 gene A 416G SNP site and the sequence of forming by SEQ ID NO.12 and SEQ ID NO.24.
8. according to each described CYP4F2 of claim 6-8 and EPHX1 gene SNP detection liquid-phase chip, described spacerarm sequence is 5-10 T.
9. be used for the Auele Specific Primer that the CYP4F2 gene SNP detects, it is characterized in that, include at the SEQID NO.13 in G1297A SNP site and SEQ ID NO.14 and/or at the SEQ ID NO.15 and the SEQ ID NO.16 in T34G SNP site.
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| CN103031364A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | FGFR3 gene mutation detection specific primer and liquid chip |
| CN103031368A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | FGFR1 gene mutation detection specific primer and liquid chip |
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| WO2006123943A1 (en) * | 2005-05-20 | 2006-11-23 | Synergenz Bioscience Limited | Methods of analysis of polymorphisms and uses 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 |
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| WO2006123943A1 (en) * | 2005-05-20 | 2006-11-23 | Synergenz Bioscience Limited | Methods of analysis of polymorphisms and uses 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 |
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Cited By (4)
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| CN103031364A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | FGFR3 gene mutation detection specific primer and liquid chip |
| CN103031368A (en) * | 2011-09-30 | 2013-04-10 | 广州益善生物技术有限公司 | FGFR1 gene mutation detection specific primer and liquid chip |
| CN103031364B (en) * | 2011-09-30 | 2014-06-18 | 益善生物技术股份有限公司 | FGFR3 gene mutation detection specific primer and liquid chip |
| CN103031368B (en) * | 2011-09-30 | 2014-06-18 | 益善生物技术股份有限公司 | FGFR1 gene mutation detection specific primer and liquid chip |
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| CN102021237B (en) | 2013-08-28 |
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