CN102912006B - CYP2A6 gene polymorphism detection specific primers and liquid chip - Google Patents
CYP2A6 gene polymorphism detection specific primers and liquid chip Download PDFInfo
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Abstract
The invention discloses CYP2A6 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of 5' terminal tag sequence and 3' terminal specific primer sequences aiming at target gene mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.17 and SEQ ID NO.18 aiming at G84T site; SEQ IDNO.19 and SEQ ID NO.20 aiming at G133A site; SEQ ID NO.21 and SEQ ID NO.22 aiming at T229A site; SEQ ID NO.23 and SEQ ID NO.24 aiming at T106C site; SEQ ID NO.25 and SEQ ID NO.26 aiming at G148T site; SEQ ID NO.27 and SEQ ID NO.28 aiming at T149G site; SEQ ID NO.29 and SEQ IDNO.30 aiming at T53C site; and/or SEQ ID NO.31 and SEQ ID NO.32 aiming at G108A site. The matching rate of the detection result obtained by using the liquid chip provided by the invention and a result obtained by using a sequencing method is 100%. Therefore, wild-type and mutant-type parallel detections of a plurality of mutation sites are realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of CYP2A6 gene pleiomorphism detection specificity primer and the liquid-phase chip of relating to.
Background technology
In recent years, become the hot subject in tumor research field about the research of Metabolic Enzyme Polymorphisms Versus and tumor susceptibility sexual intercourse.CYP II family is that at present known CYP isozyme, quantity is maximum, the family that structure is the most complicated, wherein CYP2A gene family between No. 19 long-armed 19q12 of karyomit(e) of human body and 19q13.2, about 350kb size.It comprises 3 complete genome: CYP2A6, CYP2A13 and CYP2A7, and two CYP2A7 pseudogenes.These genes all have the typical CYP2A gene subtribe structure containing 9 exons.Wherein CYP2A6 is most important member, coding tonka bean camphor 7-hydroxylase (eoumarin7-hydroxide), in the metabolism of many xenobioticses, play an important role, and participate in the metabolism activation of many procarcinogen generating through C-oxidation (C-oxidation) can peaceful (cotinine) process of iron, mainly enzymatic by CYP2A6, thus CYP2A6 gene and caused by smoking lung cancer closely related.
The CYP2A6 gene mutation site (pleomorphism site) of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of CYP2A6 site mutation | Write a Chinese character in simplified form |
| 1 | , there is G → T sudden change in the 84th Nucleotide of SEQ ID NO.69 | G84T |
| 2 | , there is G → A sudden change in the 133rd Nucleotide of SEQ ID NO.70 | G133A |
| 3 | , there is T → A sudden change in the 229th Nucleotide of SEQ ID NO.70 | T229A |
| 4 | , there is T → C sudden change in the 106th Nucleotide of SEQ ID NO.71 | T106C |
| 5 | , there is G → T sudden change in the 148th Nucleotide of SEQ ID NO.71 | G148T |
| 6 | , there is T → G sudden change in the 149th Nucleotide of SEQ ID NO.72 | T149G |
| 7 | , there is T → C sudden change in the 53rd Nucleotide of SEQ ID NO.73 | T53C |
| 8 | , there is G → A sudden change in the 108th Nucleotide of SEQ ID NO.74 | G108A |
At present, the method that CYP2A6 gene pleiomorphism is detected, analyzed mainly contains: direct sequencing and PCR-RFLP analytical method etc., wherein the most frequently used method has PCR-RFLP analytical method.PCR-RFLP method is the change of the restriction enzyme enzyme recognition site that causes based on transgenation, as lost or generation novel site in site, by a certain specific fragment of pcr amplification, use again digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, the transgenation that this method changes for detection of restriction enzyme site, can directly judge genotype, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Again, above these methods all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not meet the needs of practical application.
Summary of the invention
One of object of the present invention is to provide CYP2A6 gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the saltant type of CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A.
Realize above-mentioned purpose technical scheme as follows:
A kind of CYP2A6 gene pleiomorphism detects liquid-phase chip, includes:
(A). the wild-type designing respectively for the different pleomorphism sites of CYP2A6 gene and the ASPE primer pair of saltant type: every ASPE primer is made up of for the specific primer sequence of goal gene pleomorphism site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; For SEQ ID NO.19 and the SEQ ID NO.20 in G133A site; For SEQ ID NO.21 and the SEQ ID NO.22 in T229A site; For SEQ ID NO.23 and the SEQ ID NO.24 in T106C site; For SEQ ID NO.25 and the SEQ ID NO.26 in G148T site; For SEQ ID NO.27 and the SEQ ID NO.28 in T149G site; For SEQ ID NO.29 and the SEQ IDNO.30 in T53C site; And/or for SEQ ID NO.31 and the SEQ ID NO.32 in G108A site; Described tag sequence is selected from SEQ IDNO.1～SEQ ID NO.16;
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.33～SEQ ID NO.48, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, there is corresponding pleomorphism site.
Described amplimer is: for SEQ IDNO.49 and the SEQ IDNO.50 in G84T site; For SEQ ID NO.51 and the SEQ ID NO.52 in G133A, T229A site; For SEQ ID NO.53 and the SEQ IDNO.54 in T106C, G148T site; For SEQ ID NO.55 and the SEQ ID NO.56 in T149G site; For SEQ ID NO.57 and the SEQ ID NO.58 in T53C site; And/or for SEQ ID NO.59 and the SEQ ID NO.60 in G108A site.
Described ASPE primer is: the sequence forming for the sequence being made up of SEQ IDNO.1 and SEQ IDNO.17 in G84T site and by SEQ ID NO.2 and SEQ ID NO.18; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.19 in G133A site and the sequence that formed by SEQ ID NO.4 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.21 in T229A site and the sequence that formed by SEQ ID NO.6 and SEQ ID NO.22; For the sequence being formed by SEQ ID NO.7 and SEQ ID NO.23 in T106C site and the sequence that formed by SEQ ID NO.8 and SEQ ID NO.24; For the sequence being formed by SEQ ID NO.9 and SEQ ID NO.25 in G148T site and the sequence being formed by SEQ ID NO.10 and SEQ IDNO.26; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.27 in T149G site and the sequence that formed by SEQID NO.12 and SEQ ID NO.28; For the sequence being formed by SEQ ID NO.13 and SEQ ID NO.29 in T53C site and the sequence that formed by SEQ ID NO.14 and SEQ ID NO.30; And/or for the sequence being formed by SEQ IDNO.15 and SEQ ID NO.31 in G108A site and the sequence that formed by SEQ ID NO.16 and SEQ ID NO.32.
Another object of the present invention is to provide a kind of Auele Specific Primer detecting for CYP2A6 gene pleiomorphism.
Realize this object technical scheme as follows:
The Auele Specific Primer detecting for CYP2A6 gene pleiomorphism, described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; For SEQ ID NO.19 and the SEQ ID NO.20 in G133A site; For SEQ ID NO.21 and the SEQ ID NO.22 in T229A site; For SEQ ID NO.23 and the SEQ IDNO.24 in T106C site; For SEQ ID NO.25 and the SEQ ID NO.26 in G148T site; For SEQ ID NO.27 and the SEQ ID NO.28 in T149G site; For SEQ ID NO.29 and the SEQ ID NO.30 in T53C site; And/or for SEQ ID NO.31 and the SEQ ID NO.32 in G108A site.
Major advantage of the present invention is:
1. the identical rate of the detected result of detection liquid-phase chip provided by the present invention and sequencing is up to 100%, and detects the needed time well below conventional sequencing technologies, and realistic especially application needs.Prepared CYP2A6 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and between designed probe and anti-tag sequence, substantially there is not cross reaction, choosing and tag sequence label and the specifically combination of ASPE primer of tag sequence label, anti-tag sequence label, can avoid cross reaction, realize the parallel detection of multiple pleomorphism sites.
2. the present invention, by the design experiences of the long-term accumulation of contriver and a large amount of experimental implementation, has chosen optimum combination from numerous Auele Specific Primers.The ASPE primer specificity primer of the present invention design can the sensitive mutational site of identifying specifically target detect, accurately distinguishes the genotype of various types; In same reaction system, between different Auele Specific Primers, substantially there is not cross reaction between Auele Specific Primer and the pcr amplification product of non-target detect, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting Single locus sudden change situation, the also polymorphism situation in the multiple mutational sites of parallel detection simultaneously, detects effect consistent.
3. detection method step of the present invention is simple, eight kinds of pleomorphism sites detect the amplification that can complete by a step multiplex PCR six target sequences that contain pleomorphism site, the many uncertain factors that exist in the complex operations processes such as repeated multiple times PCR are avoided, thereby can greatly improve Detection accuracy, embody accurate while qualitative and quantitative analysis feature.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the poor defect of repeatability 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 detecting improves greatly, thereby the sensitivity detecting is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the technical scheme that the CYP2A6 gene mutation detection liquid-phase chip in the present invention provides a kind of difference to conceive for the parallel detection of multiple sites Multi-genotype, and produced significant technique effect.Meanwhile, due to the technical characterictics such as high-throughput high specific, the demand of more realistic application.Liquid-phase chip technology of the present invention is by the technology trends that is representing that biological target detects.
Embodiment
Embodiment 1CYP2A6 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
For wild-type and the saltant type of CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A, design respectively specific primer sequence.ASPE primer is made up of " Tag sequence+specific primer sequence ".ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1CYP2A6 gene
Every ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer fragment (as shown in Table 1 above) of wild-type.All ASPE primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Two, the coated microballoon of anti-tag sequence
According to designed ASPE Auele Specific Primer fragment, select tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE Auele Specific Primer fragment may form, on 16 kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 2:
Corresponding anti-tag sequence on table 2 microballoon numbering and microballoon
16 kinds of microballoons selecting, purchased from Luminex company of the U.S., are coated in anti-tag sequence on microballoon.Between anti-tag sequence and microballoon, be connected with the spacerarm sequence of 5-10 T, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for by anti-tag and microsphere surface is spaced apart or anti-tag is placed in to 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 efficiency 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), 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 coated process of microballoon is as follows:
Get respectively 5 × 10
6the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/ml) of 10ul.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (purchased from the Pierce Chemical company) working fluid of preparation 10ng/ml.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.The Tris-EDTA solution [10mmol/L Tris (pH8.0)] that the microballoon that is coated with anti-tag sequence after washing is resuspended in to 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A, design of amplification primers, to (in table 3), amplifies respectively six target sequences that contain pleomorphism site.
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Embodiment 2 uses the CYP2A6 gene pleiomorphism described in embodiment 1 to detect the detection of liquid-phase chip to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 × Tm hybridization buffer
| Reagent | 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 |
After filtration, be stored in 4 ℃.
ExoSAP-IT test kit is purchased from USB company of the U.S..
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Methods involving with reference to " molecular cloning " about DNA extraction, obtains DNA to be detected.
Two, the pcr amplification of testing sample
Design six pairs of primers, multiplex PCR one step amplifies the six objective sequences that contain respectively CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A, wherein, G133A and T229A are positioned at same amplified production, T106C and G148T and are positioned at same amplified production, product size is respectively 247bp, 391bp, 282bp, 334bp, 173bp and 336bp, and primer sequence (SEQ ID NO.49-60) is shown in shown in above-mentioned table 3.
First prepare multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.49-60 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
Pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ save backup.
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;
Hatch 15min for 2.37 ℃, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.Enzyme is cut product after treatment and 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 multiple biotin labeling on reacted product band.
First the ASPE primer working fluid that preparation mixes: get respectively the corresponding wild-type of gene to be detected and saltant type ASPE primer stock solution 10ul in 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer to mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization
1. according to the ASPE primer of design, every kind of microballoon concentration of the corresponding 16 kinds of microballoons of every group selection (as described in Example 1) is 2.5 × 10
5individual/ml.Every kind of microballoon is encoded with different colours respectively, while, every kind of microsphere surface was connected with respectively the specific oligonucleotide sequence (anti-tag) of one section of 24bp, the tag sequence specific combination that these anti-tag sequences can be held with corresponding ASPE primer 5 ' respectively;
2. get respectively the microballoon of every kind of numbering of 1ul 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, and vortex mixes;
5. get the above-mentioned microballoon suspension of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul
2o;
6. get the ASPE reaction solution of 5-25ul in corresponding hole, use ddH
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 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 the SA-PE (SA-PE) of 10ug/ml;
Hatch 15min for 12.37 ℃, on Luminex instrument, detect.
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.Detected result is as shown in table 4, table 5 and table 6.Fluorescent value (MFI) and data processing are had to following requirement:
1. each site need have at least an allelotrope MFI to be greater than 300 and be greater than 10 × PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI be less than 0 represent with 0);
3. meet the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NETMFI+ wild-type NET MFI)
4. the sudden change ratio definite threshold (cut-off) to each detection site rule of thumb, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments CYP2A6 gene polymorphism sites originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Arranging of threshold value (cut-off value) is as follows: sudden change ratio range is considered as wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as anomaly homozygote.Detect with liquid-phase chip result and compare with sequencing, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments CYP2A6 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible CYP2A6 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect CYP2A6 gene polymorphism sites type exactly, and result is reliable and stable.
One of table 4 pattern detection result (MFI)
Two of table 5 pattern detection result (MFI)
Table 6 sample CYP2A6 transgenation ratio (%)
Table 7 sample CYP2A6 gene mutation type analytical results
The detection of the liquid-phase chip of the ASPE primer that embodiment 3 is different to CYP2A6 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example take CYP2A6 gene T106C and T149G site mutation, respectively for the wild-type of T106C and T149G and the specific primer sequence of saltant type design ASPE primer 3 ' end, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.1-SEQ ID NO.16, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.33-SEQ ID NO.48.Specific design is as shown in following table (table 8).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 8 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 21-40 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 9 pattern detection result and Polymorphism Analysis
Table 10 pattern detection result and Polymorphism Analysis
Other is for the liquid-phase chip in different mutational sites, and ASPE primer uses different tag sequences, and its result is still reliable and stable, and concrete data are omitted.And ASPE primer is while selecting in embodiment 1 collocation of tag sequence and specific primer sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 2 and test group 5.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
The selection of embodiment 4CYP2A6 gene pleiomorphism detection specificity primer sequence
One, the design (selection of wild-type and saltant type specific primer sequence) that prepared by liquid-phase chip
Detect liquid-phase chip as example take the pleomorphism site of CYP2A6 gene T229A and G148T, take the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for the wild-type of T229A and G148T and the specific primer sequence of saltant type design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the present invention 1, as shown in table 11.Wherein,
interior base is pleomorphism site.
Table 11 specific primer sequence
Detect liquid-phase chip as example take the pleomorphism site of CYP2A6 gene T229A and G148T, select different specific primer sequences for T229A and G148T, the Tag sequence of ASPE primer 5 ' end is fixed as the best effect sequence in embodiment 1, and select the anti-tag sequence of answering in contrast, specific design is as shown in following table (table 12).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Two of design prepared by table 12 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 41-60 by testing process described in embodiment 2 and method, and detected result is as follows:
Table 13 pattern detection result and Polymorphism Analysis
Table 14 pattern detection result and Polymorphism Analysis
From the present embodiment, when ASPE primer is selected in embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 7 and test group 10.Other derives from different specific primer sequences and the collocation of tag sequence of the complementary sequence forward or backwards of place, target detect site sequence, with coming to the same thing of embodiment 2 and the present embodiment, be still that the specific primer sequence described in embodiment 2 is better from different tag sequence arranging effects, concrete data are omitted.Other multiple specific primer sequence for different mutational sites and the collocation of tag sequence, with coming to the same thing of embodiment 2 and the present embodiment, the selected Auele Specific Primer of embodiment 1, has better signal to noise ratio, detect effect also better, concrete data are omitted.
Be more than for the illustrating of possible embodiments of the present invention, but this embodiment is not in order to limit the scope of the claims of the present invention, allly do not depart from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the scope of the claims of the present invention.
Claims (6)
1. CYP2A6 gene pleiomorphism detects a liquid-phase chip, it is characterized in that, includes:
(A). the wild-type designing respectively for the different pleomorphism sites of CYP2A6 gene and the ASPE primer pair of saltant type: every ASPE primer is made up of for the specific primer sequence of goal gene pleomorphism site tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; For SEQ ID NO.19 and the SEQ ID NO.20 in G133A site; For SEQ ID NO.21 and the SEQ ID NO.22 in T229A site; For SEQ ID NO.23 and the SEQ ID NO.24 in T106C site; For SEQ ID NO.25 and the SEQ ID NO.26 in G148T site; For SEQ ID NO.27 and the SEQ ID NO.28 in T149G site; For SEQ ID NO.29 and the SEQ IDNO.30 in T53C site; And/or for SEQ ID NO.31 and the SEQ ID NO.32 in G108A site; Described tag sequence is selected from SEQ IDNO.1～SEQ ID NO.16:
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.33～SEQ ID NO.48, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). for amplifying the primer that needs the target sequence detecting, there is corresponding pleomorphism site.
2. CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1, it is characterized in that, described amplimer is: for SEQ ID NO.49 and the SEQ ID NO.50 in G84T site; For SEQ ID NO.51 and the SEQ ID NO.52 in G133A, T229A site; For SEQ ID NO.53 and the SEQ ID NO.54 in T106C, G148T site; For SEQ ID NO.55 and the SEQ ID NO.56 in T149G site; For SEQ ID NO.57 and the SEQ ID NO.58 in T53C site; And/or for SEQ ID NO.59 and the SEQ ID NO.60 in G108A site.
3. CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: for the sequence being made up of SEQ ID NO.1 and SEQ ID NO.17 in G84T site and the sequence being made up of SEQ ID NO.2 and SEQ IDNO.18; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.19 in G133A site and the sequence that formed by SEQ IDNO.4 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.21 in T229A site and the sequence that formed by SEQ ID NO.6 and SEQ ID NO.22; The sequence forming for the sequence being formed by SEQ ID NO.7 and SEQID NO.23 in T106C site and by SEQ ID NO.8 and SEQ ID NO.24; For the sequence being formed by SEQID NO.9 and SEQ ID NO.25 in G148T site and the sequence that formed by SEQ ID NO.10 and SEQ ID NO.26; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.27 in T149G site and the sequence being formed by SEQ ID NO.12 and SEQ IDNO.28; For the sequence being formed by SEQ ID NO.13 and SEQ ID NO.29 in T53C site and the sequence that formed by SEQ IDNO.14 and SEQ ID NO.30; And/or for the sequence being formed by SEQ ID NO.15 and SEQ ID NO.31 in G108A site and the sequence that formed by SEQ ID NO.16 and SEQ ID NO.32.
4. CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1, it is characterized in that,
(A) .ASPE primer is: for the sequence being made up of SEQ ID NO.1 and SEQ ID NO.17 in G84T site and the sequence that is made up of SEQID NO.2 and SEQ ID NO.18; For the sequence being formed by SEQ ID NO.3 and SEQ ID NO.19 in G133A site and the sequence that formed by SEQ ID NO.4 and SEQ ID NO.20; For the sequence being formed by SEQ ID NO.5 and SEQ ID NO.21 in T229A site and the sequence that formed by SEQ ID NO.6 and SEQ ID NO.22; For the sequence being formed by SEQ ID NO.7 and SEQ ID NO.23 in T106C site and the sequence that formed by SEQ ID NO.8 and SEQ ID NO.24; For the sequence being formed by SEQ ID NO.9 and SEQ ID NO.25 in G148T site and the sequence that formed by SEQ ID NO.10 and SEQ ID NO.26; For the sequence being formed by SEQ ID NO.11 and SEQ ID NO.27 in T149G site and the sequence that formed by SEQ IDNO.12 and SEQ ID NO.28; For the sequence being formed by SEQ ID NO.13 and SEQ ID NO.29 in T53C site and the sequence that formed by SEQ ID NO.14 and SEQ ID NO.30; With the sequence being formed by SEQ ID NO.15 and SEQ ID NO.31 for G108A site and the sequence being formed by SEQ ID NO.16 and SEQ ID NO.32;
(B). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.33～SEQ ID NO.48, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). described amplimer is: for SEQ ID NO.49 and the SEQ ID NO.50 in G84T site; For SEQ ID NO.51 and the SEQ ID NO.52 in G133A, T229A site; For SEQ ID NO.53 and the SEQID NO.54 in T106C, G148T site; For SEQ ID NO.55 and the SEQ ID NO.56 in T149G site; For SEQ ID NO.57 and the SEQ ID NO.58 in T53C site; With SEQ ID NO.59 and the SEQ ID NO.60 for G108A site.
5. detect liquid-phase chip according to the CYP2A6 gene pleiomorphism described in claim 1-4 any one, it is characterized in that, described spacerarm is 5-10 T.
6. the Auele Specific Primer detecting for CYP2A6 gene pleiomorphism, is characterized in that, described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; For SEQ ID NO.19 and the SEQ IDNO.20 in G133A site; For SEQ ID NO.21 and the SEQ ID NO.22 in T229A site; For SEQ ID NO.23 and the SEQ ID NO.24 in T106C site; For SEQ ID NO.25 and the SEQ ID NO.26 in G148T site; For SEQID NO.27 and the SEQ ID NO.28 in T149G site; For SEQ ID NO.29 and the SEQ ID NO.30 in T53C site; And/or for SEQ ID NO.31 and the SEQ ID NO.32 in G108A site.
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| 童紫莺等.细胞色素P4502A6的多态性研究.《中华医学遗传学杂志》.2002,(第05期), |
| 细胞色素P4502A6的多态性研究;童紫莺等;《中华医学遗传学杂志》;20021026(第05期);表1 * |
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