CN102912006A - 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 quantity is maximum in the present known CYP isozyme, the family that structure is the most complicated, and wherein the CYP2A gene family is between No. 19 long-armed 19q12 of karyomit(e) of human body and 19q13.2, and about 350kb is big or small.It comprises 3 complete genome: CYP2A6, CYP2A13 and CYP2A7, and two CYP2A7 pseudogenes.These genes all have the typical CYP2A gene subtribe structure that contains 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 through C-oxidation (C-oxidation) but generate peaceful (cotinine) process of iron, mainly be enzymatic by CYP2A6, thereby CYP2A6 gene and caused by smoking lung cancer are 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 | G → T sudden change occurs in the 84th Nucleotide of SEQ ID NO.69 | G84T |
| 2 | G → A sudden change occurs in the 133rd Nucleotide of SEQ ID NO.70 | G133A |
| 3 | T → A sudden change occurs in the 229th Nucleotide of SEQ ID NO.70 | T229A |
| 4 | T → C sudden change occurs in the 106th Nucleotide of SEQ ID NO.71 | T106C |
| 5 | G → T sudden change occurs in the 148th Nucleotide of SEQ ID NO.71 | G148T |
| 6 | T → G sudden change occurs in the 149th Nucleotide of SEQ ID NO.72 | T149G |
| 7 | T → C sudden change occurs in the 53rd Nucleotide of SEQ ID NO.73 | T53C |
| 8 | G → A sudden change occurs in the 108th Nucleotide of SEQ ID NO.74 | G108A |
At present, the method that the CYP2A6 gene pleiomorphism is detected, analyzes mainly contains: direct sequencing and PCR-RFLP analytical method etc., wherein the most frequently used method has the PCR-RFLP analytical method.The PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes, as losing or produce novel site in the site, by a certain specific fragment of pcr amplification, use again the digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, this method can directly be judged genotype for detection of the transgenation that restriction enzyme site changes, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Again, more than these methods all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
Summary of the invention
One of purpose of the present invention provides the CYP2A6 gene pleiomorphism and detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A.
Realize that the above-mentioned purpose technical scheme is as follows:
A kind of CYP2A6 gene pleiomorphism detects liquid-phase chip, includes:
(A). the wild-type that designs respectively for the different pleomorphism sites of CYP2A6 gene and the ASPE primer of mutant pair: every ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene pleomorphism site, and described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; SEQ ID NO.19 and SEQ ID NO.20 for the G133A site; SEQ ID NO.21 and SEQ ID NO.22 for the T229A site; SEQ ID NO.23 and SEQ ID NO.24 for the T106C site; SEQ ID NO.25 and SEQ ID NO.26 for the G148T site; SEQ ID NO.27 and SEQ ID NO.28 for the T149G site; SEQ ID NO.29 and SEQ IDNO.30 for the 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). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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). be used for amplifying the primer that needs target sequence detection, that have corresponding pleomorphism site.
Described amplimer is: for SEQ IDNO.49 and the SEQ IDNO.50 in G84T site; SEQ ID NO.51 and SEQ ID NO.52 for G133A, T229A site; SEQ ID NO.53 and SEQ IDNO.54 for T106C, G148T site; SEQ ID NO.55 and SEQ ID NO.56 for the T149G site; SEQ ID NO.57 and SEQ ID NO.58 for the T53C site; And/or for SEQ ID NO.59 and the SEQ ID NO.60 in G108A site.
Described ASPE primer is: the sequence that is comprised of SEQ IDNO.1 and SEQ IDNO.17 for the G84T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.18; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.19 for the G133A site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.20; The sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.21 for the T229A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.22; The sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.23 for the T106C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.24; The sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.25 for the G148T site reaches the sequence that is comprised of SEQ ID NO.10 and SEQ IDNO.26; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.27 for the T149G site reaches the sequence that is comprised of SEQID NO.12 and SEQ ID NO.28; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.29 for the T53C site reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.30; And/or for the sequence that is formed by SEQ IDNO.15 and SEQ ID NO.31 in G108A site and the sequence that is formed by SEQ ID NO.16 and SEQ ID NO.32.
Another object of the present invention provides a kind of Auele Specific Primer for the detection of CYP2A6 gene pleiomorphism.
Realize that this purpose technical scheme is as follows:
A kind of Auele Specific Primer for the detection of CYP2A6 gene pleiomorphism, described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; SEQ ID NO.19 and SEQ ID NO.20 for the G133A site; SEQ ID NO.21 and SEQ ID NO.22 for the T229A site; SEQ ID NO.23 and SEQ IDNO.24 for the T106C site; SEQ ID NO.25 and SEQ ID NO.26 for the G148T site; SEQ ID NO.27 and SEQ ID NO.28 for the T149G site; SEQ ID NO.29 and SEQ ID NO.30 for the 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 sequencing technologies commonly used, and realistic especially application needs.Prepared CYP2A6 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and basically there is not cross reaction between designed probe and the anti-tag sequence, 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 a plurality of pleomorphism sites.
2. the present invention has chosen optimum combination by the design experiences of contriver's long-term accumulation and a large amount of experimental implementation from numerous Auele Specific Primers.The ASPE primer specificity primer of the present invention's design can sensitive be identified the mutational site of target detect specifically, accurately distinguishes the genotype of various types; In same reaction system, between the different Auele Specific Primers, basically do not have cross reaction between the pcr amplification product of Auele Specific Primer and 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 a plurality of mutational sites of simultaneously parallel detection, it is consistent to detect effect.
3. detection method step of the present invention is simple, eight kinds of pleomorphism sites detect and can finish six amplifications that contain the target sequence of pleomorphism 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 greatly improve Detection accuracy, embodied 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 defective that the repeatability of detected result is poor is improved existing liquid-phase chip technology simultaneously, so that prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby so that the sensitivity that detects is further enhanced, signal to noise ratio strengthens, and detected result more accurately and reliably.
5. the CYP2A6 gene mutation detection liquid-phase chip among the present invention provides a kind of technical scheme of different designs for the parallel detection of a plurality of sites Multi-genotype, and has produced significant technique effect.Simultaneously, since the technical characterictics such as high-throughput high specific, the demand of more realistic application.Liquid-phase chip technology of the present invention will represent the technology trends that biological target detects.
Embodiment
Embodiment 1CYP2A6 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Wild-type and mutant for CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A design respectively specific primer sequence.The ASPE primer is comprised of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1CYP2A6 gene
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (as shown in Table 1 above) 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 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 the 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, corresponding anti-tag sequence is as shown in table 2 on 16 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
16 kinds of microballoons selecting are coated in the anti-tag sequence on the microballoon available from U.S. Luminex company.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, namely 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 for being 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 such 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 that microballoon is coated with is as follows:
Get respectively 5 * 10
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 the synthetic anti-tag molecule (100nmol/ml) of 10ul.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid that adds 2.5ul in the microballoon suspension, constant-temperature incubation 30 minutes adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 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/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For CYP2A6 gene eight kinds of common genotype G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A, design of amplification primers amplifies respectively six target sequences that contain pleomorphism site to (seeing Table 3).
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthetic 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 embodiment 1 described CYP2A6 gene pleiomorphism to detect liquid-phase chip to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 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 about DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Design six pairs of primers, one step of multiplex PCR 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 the same amplified production, the product size is respectively 247bp, 391bp, 282bp, 334bp, 173bp and 336bp, and primer sequence (SEQ ID NO.49-60) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.49-60 in the 1.5ml Eppendorf tube, mix and be the multiple PCR primer working fluid.The multi-PRC reaction system is as follows:
The 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;
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 the ASPE primer working fluid that mixes: get respectively the corresponding wild-type of gene to be detected and mutant ASPE primer stock solution 10ul in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and 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, simultaneously every kind of microsphere surface is connected with respectively the specific oligonucleotide sequence (anti-tag) of one section 24bp, and these anti-tag sequences can be respectively and the tag sequence specific combination of corresponding ASPE primer 5 ' end;
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, 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. 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 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 the SA-PE (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 detects by Luminex serial analysis instrument.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 NETMFI+ wild-type NET MFI)
4. rule of thumb to the sudden change ratio definite threshold (cut-off) of each detection site, to divide wild-type homozygote, heterozygote and mutant 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: 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 CYP2A6 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen CYP2A6 gene pleiomorphism provided by the present invention detects liquid-phase chip and can detect exactly CYP2A6 gene polymorphism sites type, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 pattern detection result's (MFI) two
Table 6 sample CYP2A6 transgenation ratio (%)
Table 7 sample CYP2A6 gene mutation type analytical results
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of CYP2A6 gene polymorphism sites
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
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 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.16, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.33-SEQ ID NO.48.Specific design is shown in following table (table 8).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
The design of table 8 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 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 the ASPE primer uses different tag sequences, and its result is still reliable and stable, and concrete data are omitted.And the ASPE primer is when selecting among the embodiment 1 collocation of tag sequence and specific primer sequence, and effect better (signal to noise ratio is better) is referring to 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 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 mutant specific primer sequence) of liquid-phase chip preparation
Pleomorphism site take CYP2A6 gene T229A and G148T detects liquid-phase chip as example, take the forward or backwards complementary sequence of this place, mutational site target sequence as template, respectively for the wild-type of T229A and G148T and the specific primer sequence of mutant design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the invention 1, as shown in table 11.Wherein,
Interior base is pleomorphism site.
Table 11 specific primer sequence
Pleomorphism site take CYP2A6 gene T229A and G148T detects liquid-phase chip as example, select different specific primer sequences for T229A and G148T, the Tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1, and select with it corresponding anti-tag sequence, specific design is shown in following table (table 12).Synthetic, the coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like embodiment 1 and embodiment 2.
Two of the design of table 12 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 13 pattern detection result and Polymorphism Analysis
Table 14 pattern detection result and Polymorphism Analysis
By present embodiment as seen, when the ASPE primer was selected among the embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better) was referring to present embodiment test group 7 and test group 10.Other derives from different specific primer sequences and the collocation of tag sequence of the forward or backwards complementary sequence of place, target detect site sequence, with coming to the same thing of embodiment 2 and present embodiment, namely still be that the specific primer sequence described in the embodiment 2 is better from different tag sequence arranging effects, concrete data are omitted.Other is for multiple specific primer sequence and the collocation of tag sequence in different mutational sites, with coming to the same thing of embodiment 2 and present embodiment, namely embodiment 1 selected Auele Specific Primer has better signal to noise ratio, it is also better to detect effect, and concrete data are omitted.
More than be for the specifying of possible embodiments of the present invention, but this embodiment limits claim of the present invention, allly do not break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (6)
1. a CYP2A6 gene pleiomorphism detects liquid-phase chip, it is characterized in that, includes:
(A). the wild-type that designs respectively for the different pleomorphism sites of CYP2A6 gene and the ASPE primer of mutant pair: every ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for the goal gene pleomorphism site, and described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; SEQ ID NO.19 and SEQ ID NO.20 for the G133A site; SEQ ID NO.21 and SEQ ID NO.22 for the T229A site; SEQ ID NO.23 and SEQ ID NO.24 for the T106C site; SEQ ID NO.25 and SEQ ID NO.26 for the G148T site; SEQ ID NO.27 and SEQ ID NO.28 for the T149G site; SEQ ID NO.29 and SEQ IDNO.30 for the 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). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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). be used for amplifying the primer that needs target sequence detection, that have corresponding pleomorphism site.
2. described 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; SEQ ID NO.51 and SEQ ID NO.52 for G133A, T229A site; SEQ ID NO.53 and SEQ ID NO.54 for T106C, G148T site; SEQ ID NO.55 and SEQ ID NO.56 for the T149G site; SEQ ID NO.57 and SEQ ID NO.58 for the T53C site; And/or for SEQ ID NO.59 and the SEQ ID NO.60 in G108A site.
3. described CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1, it is characterized in that described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.17 for the G84T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ IDNO.18; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.19 for the G133A site reaches the sequence that is comprised of SEQ IDNO.4 and SEQ ID NO.20; The sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.21 for the T229A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.22; The sequence that is comprised of SEQ ID NO.7 and SEQID NO.23 for the T106C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.24; The sequence that is comprised of SEQID NO.9 and SEQ ID NO.25 for the G148T site reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.26; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.27 for the T149G site reaches the sequence that is comprised of SEQ ID NO.12 and SEQ IDNO.28; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.29 for the T53C site reaches the sequence that is comprised of SEQ IDNO.14 and SEQ ID NO.30; And/or for the sequence that is formed by SEQ ID NO.15 and SEQ ID NO.31 in G108A site and the sequence that is formed by SEQ ID NO.16 and SEQ ID NO.32.
4. described CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1, it is characterized in that,
(A) the .ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.17 for the G84T site reaches the sequence that is comprised of SEQID NO.2 and SEQ ID NO.18; The sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.19 for the G133A site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.20; The sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.21 for the T229A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.22; The sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.23 for the T106C site reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.24; The sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.25 for the G148T site reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.26; The sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.27 for the T149G site reaches the sequence that is comprised of SEQ IDNO.12 and SEQ ID NO.28; The sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.29 for the T53C site reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.30; Reach the sequence that is formed by SEQ ID NO.16 and SEQ ID NO.32 with the sequence that is formed by SEQ ID NO.15 and SEQ ID NO.31 for the G108A site;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; 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; SEQ ID NO.51 and SEQ ID NO.52 for G133A, T229A site; SEQ ID NO.53 and SEQID NO.54 for T106C, G148T site; SEQ ID NO.55 and SEQ ID NO.56 for the T149G site; SEQ ID NO.57 and SEQ ID NO.58 for the T53C site; With SEQ ID NO.59 and the SEQ ID NO.60 for the G108A site.
5. each described CYP2A6 gene pleiomorphism detects liquid-phase chip according to claim 1-4, it is characterized in that described spacerarm is 5-10 T.
6. one kind is used for the Auele Specific Primer that the CYP2A6 gene pleiomorphism detects, and it is characterized in that described specific primer sequence is: for SEQ ID NO.17 and the SEQ ID NO.18 in G84T site; SEQ ID NO.19 and SEQ IDNO.20 for the G133A site; SEQ ID NO.21 and SEQ ID NO.22 for the T229A site; SEQ ID NO.23 and SEQ ID NO.24 for the T106C site; SEQ ID NO.25 and SEQ ID NO.26 for the G148T site; SEQID NO.27 and SEQ ID NO.28 for the T149G site; SEQ ID NO.29 and SEQ ID NO.30 for the T53C site; And/or for SEQ ID NO.31 and the SEQ ID NO.32 in G108A site.
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| CN101487052A (en) * | 2009-02-24 | 2009-07-22 | 广州益善生物技术有限公司 | Liquid phase chip for CYP2C9 and CYP2C19 gene mutation detection and detecting method thereof |
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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》 * |
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