CN102226219B - Specific primer and liquid-phase chip for polymorphism detection in chromosome 6q23 section - Google Patents
Specific primer and liquid-phase chip for polymorphism detection in chromosome 6q23 section Download PDFInfo
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Abstract
The invention discloses a specific primer and a liquid-phase chip for polymorphism detection in a chromosome 6q23 section. The liquid-phase chip mainly comprises a specific primer, microspheres and amplified primers, wherein the specific primer comprises a tag sequence of a 5' terminal and a specific primer sequence of a 3' terminal for target gene polymorphism sites, wherein the specific primer is more than one pair of SEQ ID NO.15 and SEQ ID NO.16 for G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, and SEQ ID NO.27 and SEQ ID NO.28 for T135C; and the tag sequence is a sequence of SEQ ID NO.1 to SEQ ID NO.14. The prepared liquid-phase chip for polymorphism detection has a good signal-noise ratio, and the designed probe and an anti-tag sequence do not have cross reaction basically.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of karyomit(e) 6q23 section polymorphic detection Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is that a kind of inflammatory is invaded profit joint and the systemic autoimmune disease of reticular tissue on every side thereof, is also a kind of disease of multifactorial inheritance with complex inheritance pattern.Generation, the development of environmental factors and inherited genetic factors joint effect RA.
At present, study (genome wideassociations study by whole-genome association, GWAS) show, the tumor susceptibility gene relevant to RA mainly contains HLA (human leucocyte antigen) gene that is positioned at 6p21.3, PTPN22 (the non-receptor protein tyrosine phosphatase 22) gene that is positioned at 1p23,6q23 site, CTLA-4 (cytotoxic lymphocyte antigen 4) gene, 9q33-34 site (comprising two genes of TRAF1 and C5), is positioned at No. 2 chromosomal STAT4 (signal transduction and transcription activator 4) gene etc.
Wherein, the present invention is mainly for the 6q23 site with OLIG3 (oligodendrocyte lineage transcription factor 3) and TNFAIP3 (TNF-a-induced protein 3) gene vicinity, and the function of TNFAIP3 is by lowering its inflammatory reaction caused of nf NF-κ B concentration negative regulation.The sudden change of 6q23 section related locus will affect the expression of TNFAIP3 gene product, thereby affect the occurrence risk of RA.
The karyomit(e) 6q23 block mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of karyomit(e) 6q23 section site mutation | Write a Chinese character in simplified form |
| 1 | G → A sudden change, occur in the 107th Nucleotide of SEQ ID NO.57 | G107A |
| 2 | G → A sudden change, occur in the 301st Nucleotide of SEQ ID NO.58 | G301A |
| 3 | G → A sudden change, occur in the 103rd Nucleotide of SEQ ID NO.59 | G103A |
| 4 | G → T sudden change, occur in the 219th Nucleotide of SEQ ID NO.60 | G219T |
| 5 | T → C sudden change, occur in the 74th Nucleotide of SEQ ID NO.61 | T74C |
| 6 | T → C sudden change, occur in the 163rd Nucleotide of SEQ ID NO.62 | T163C |
| 7 | T → C sudden change, occur in the 135th Nucleotide of SEQ ID NO.63 | T135C |
At present, the method that karyomit(e) 6q23 section gene pleiomorphism is detected, analyzes is few, generally has: traditional solid phase chip and TaqMan technology etc., wherein the most frequently used method is based on the TaqMan technical process of quantitative fluorescent PCR.Easy and simple to handle, the advantages such as result quick, quantification that the TaqMan technology has, but, this technology exists sample easily to pollute, cross reaction easily occurs, the shortcoming that false positive rate is high, simultaneously, the method exists the limitation that detects flux, a kind of mutation type can only be detected at every turn, the needs of practical application can not be met.
Summary of the invention
One of purpose of the present invention is to provide karyomit(e) 6q23 section polymorphic detection liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the saltant type of 7 kinds of common genotype G107A, G301A, G103A, G219T, T74C, T163C and T135C of karyomit(e) 6q23 section.
A kind of karyomit(e) 6q23 section polymorphic detection liquid-phase chip mainly includes:
A. the wild-type designed respectively for 7 kinds of common polymorphisms sites of karyomit(e) 6q23 section and saltant type specificity ASPE primer, every kind of ASPE primer is comprised of the tag sequence of 5 ' end and the specific primer sequence for the goal gene pleomorphism site of 3 ' end, described specific primer sequence is selected from respectively: for SEQ ID NO.15 and the SEQ ID NO.16 of G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ IDNO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, for more than a pair of in the SEQ ID NO.27 of T135C and SEQ ID NO.28, described tag sequence is selected from the sequence in SEQ ID NO.1～SEQ ID NO.14,
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in SEQ ID NO.29～SEQ ID NO.42, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. there is the primer of the above pleomorphism site target sequence of G107A, G301A, G103A, G219T, T74C and T135C for amplification.
Preferably, described amplimer is: for the SEQ ID NO.43 of G107A and SEQ ID NO.44, for the SEQID NO.45 of G301A and SEQ ID NO.46, for the SEQ ID NO.47 of G103A and SEQ ID NO.48, for the SEQ IDNO.49 of G219T and SEQ ID NO.50, for the SEQ ID NO.51 of T74C and SEQ ID NO.52, for the SEQ IDNO.53 of T163C and SEQ ID NO.54, for more than a pair of in the SEQ ID NO.55 of T135C and SEQ ID NO.56.
Preferably, described ASPE primer is: for the sequence be comprised of SEQ ID NO.1 and SEQ ID NO.15 of G107A and the sequence be comprised of SEQ ID NO.2 and SEQ ID NO.16, for the sequence formed by SEQ ID NO.3 and SEQ ID NO.17 of G301A and the sequence formed by SEQ ID NO.4 and SEQ ID NO.18, for the sequence formed by SEQ ID NO.5 and SEQ ID NO.19 of G103A and the sequence formed by SEQ ID NO.6 and SEQ ID NO.20, for the sequence formed by SEQID NO.7 and SEQ ID NO.21 of G219T and the sequence formed by SEQ ID NO.8 and SEQ ID NO.22, for the sequence formed by SEQ ID NO.9 and SEQ ID NO.23 of T74C and the sequence formed by SEQ ID NO.10 and SEQ ID NO.24, for the sequence formed by SEQ ID NO.11 and SEQ ID NO.25 of T163C and the sequence formed by SEQ ID NO.12 and SEQID NO.26, for more than a pair of in the sequence formed by SEQ ID NO.13 and SEQ ID NO.27 of T135C and the sequence that formed by SEQ IDNO.14 and SEQ ID NO.28.
Another object of the present invention is to provide a kind of specific primer sequence for karyomit(e) 6q23 section polymorphic detection.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of specific primer sequence for karyomit(e) 6q23 section polymorphic detection, it is selected from: for the SEQ IDNO.15 of G107A and SEQ ID NO.16, for the SEQ ID NO.17 of G301A and SEQ ID NO.18, for the SEQ IDNO.19 of G103A and SEQ ID NO.20, for the SEQ ID NO.21 of G219T and SEQ ID NO.22, for the SEQ IDNO.23 of T74C and SEQ ID NO.24, for the SEQ ID NO.25 of T163C and SEQ ID NO.26, for more than a pair of in the SEQ IDNO.27 of T135C and SEQ ID NO.28.
Major advantage of the present invention is:
1. the identical rate of the detected result of 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.In very a large amount of Auele Specific Primers, through lot of experiments, reaction is verified, obtains the liquid-phase chip system of optimum combination.Prepared karyomit(e) 6q23 section polymorphic detection liquid-phase chip has extraordinary signal-noise ratio, and basically there do not is cross reaction between designed probe and 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 in a plurality of SNP site.
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, between the pcr amplification product of Auele Specific Primer and non-target detect, basically do not have cross reaction, detection specificity is good, and cross reacting rate is lower than 3%; Except detecting Single locus sudden change situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously, detect effect consistent.
3. detection method step of the present invention is simple, 7 kinds of pleomorphism sites detect and can complete the amplification of 7 target sequences that contain the SNP site by a step multiplex PCR, the many uncertain factors that exist 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 defect that the repeatability of detected result is poor 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 detected improves greatly, thereby makes the sensitivity of detection be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 karyomit(e) 6q23 section polymorphic detection liquid-phase chip
Mainly include:
One, ASPE primer
Wild-type and saltant type for 7 kinds of common genotype G107A, G301A, G103A, G219T, T74C, T163C and T135C of karyomit(e) 6q23 section, 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 1 karyomit(e) 6q23 section
Every the 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 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, on 14 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
14 kinds of microballoons selecting are purchased from U.S. Luminex company, by the anti-tag sequence coated with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and microballoon, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for 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 of individual above-mentioned numbering (purchased 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) (purchased from the Pierce Chemical company) working fluid of preparation 10ng/ml.Toward the EDC working fluid that adds 2.5ul in the 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 microballoon that is coated with the anti-tag sequence after washing is resuspended in to the Tris-EDTA solution [10mmol/LTris (pH8.0)] of 100ul, in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains mutational site
For 7 kinds of common genotype G107A, G301A, G103A, G219T, T74C, T163C and T135C of karyomit(e) 6q23 section, design of amplification primers, to (in Table 3), amplifies respectively 7 target sequences that contain pleomorphism site.
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.
The detection of the applicable described karyomit(e) 6q23 of the embodiment 1 zone detection liquid-phase chip of embodiment 2 to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
| Reagent | Source | Final concentration | The consumption of every 250ml |
| MES(2[N-Morpholino] | Sigma M-2933 | 0.05M | 2.44g |
| ethanesulfonic acid) | |||
| 5MNaOH | Fisher SS256-500 | --- | 5 |
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 |
Be stored in 4 ℃ after filtration.
The ExoSAP-IT test kit is purchased from U.S. USB company.
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, obtain DNA to be detected.
Two, by the pcr amplification of test sample product
Design 7 pairs of primers, multiplex PCR one step amplifies 7 kinds of common genotype G107A, G301A, G103A, G219T, T74C, T163C and T135C totally 7 objective sequences that contain respectively karyomit(e) 6q23 section, the product size is respectively 464bp, 616bp, 351bp, 453bp, 278bp, 393bp and 345bp, and primer sequence (SEQ ID NO.43-56) is shown in shown in 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.43-56 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 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, mix biotin labeled dCTP in reaction process, thereby make a plurality of biotin labeling on reacted product band.
At first the ASPE primer working fluid that preparation mixes: get respectively the corresponding wild-type in site to be detected and saltant type ASPE primer stock solution 10ul in the 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 is 2.5 * 10 to the corresponding 28 kinds of microballoons of every group selection
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 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. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, adds the SA-PE (SA-PE) that 15ul concentration is 10ug/ml;
12.37 ℃ hatch 15min, detect on the Luminex instrument.
Six, result detects and data analysis
The 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 with 0 expression);
3. meet the data of above two conditions, by following formula, calculate sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NET MFI+ wild-type NET MFI)
4. rule of thumb to the sudden change ratio definite threshold (cut-off value) of each detection site, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments karyomit(e) 6q23 section pleomorphism site 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.Detect with the 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 karyomit(e) 6q23 sector type detected result and the sequencing result rate of coincideing originally and reaches 100%.Visible karyomit(e) 6q23 section polymorphic detection liquid-phase chip provided by the present invention can detect karyomit(e) 6q23 section polymorphic position vertex 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 dyeing body 6q23 block mutation ratio (%)
Table 7 sample dyeing body 6q23 block mutation type analysis result
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | 163CC | 163CC |
| 2 | 219GT | 219GT |
| 3 | 74CC | 74CC |
| 4 | Wild-type | Wild-type |
| 5 | 107AA | 107AA |
| 6 | Wild-type | Wild-type |
| 7 | Wild-type | Wild-type |
| 8 | 301AA、103AA | 301AA、103AA |
| 9 | 135CC | 135CC |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | 163TC | 163TC |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | 107GA、219GT | 107GA、219GT |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
The detection of the liquid-phase chip ginseng karyomit(e) 6q23 section pleomorphism site of the ASPE primer that embodiment 3 is different
One, the design that prepared by liquid-phase chip (selection of Tag sequence and Anti-Tag sequence)
Take karyomit(e) 6q23 section G301A and G219T site mutation, to detect liquid-phase chip be example, respectively for the wild-type of G301A and G219T 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.14, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing be coated on microballoon is selected from SEQ ID NO.29-SEQ ID NO.42.Specific design is as 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.
Design prepared by table 8 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detected sample 21-40 by the described testing process of 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 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 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 4 karyomit(e) 6q23 section polymorphic detection specific primer sequences
One, the design that prepared by liquid-phase chip (selection of wild-type and saltant type specific primer sequence)
Take karyomit(e) 6q23 section G103A and T163C site mutation, to detect liquid-phase chip be example, the complementary sequence forward or backwards of this place, mutational site target sequence of take is template, respectively for the wild-type of G103A and T163C 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
Take respectively karyomit(e) 6q23 section G103A and T163C site mutation, to detect liquid-phase chip be example, select different specific primer sequences for G103A and T163C, 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 coated microballoon of anti-tag sequence of ASPE primer, amplimer, detection method are described like 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, detected sample 41-60 by the described testing process of 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, can find out, when the 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 experimental group 10.The different specific primer sequences of complementary sequence forward or backwards and the collocation of tag sequence that other derives from place, target detect site sequence, be still in embodiment 1 specific primer sequence and tag sequence arranging effect better.Other 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, have better signal to noise ratio, detects effect also better, and 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 break away 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 (5)
1. a karyomit(e) 6q23 section polymorphic detection liquid-phase chip, is characterized in that, mainly includes:
A. the wild-type and the saltant type specificity ASPE primer that for the different pleomorphism sites of karyomit(e) 6q23 section, design respectively, every kind of ASPE primer is comprised of the tag sequence of 5 ' end and the specific primer sequence for the goal gene pleomorphism site of 3 ' end, described specific primer sequence is selected from respectively: for SEQ ID NO.15 and the SEQ ID NO.16 of G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, for more than a pair of in the SEQ ID NO.27 of T135C and SEQ ID NO.28, described tag sequence is selected from the sequence in SEQ ID NO.1～SEQ ID NO.14,
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in SEQ ID NO.29～SEQ ID NO.42, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. there is the primer of the above pleomorphism site target sequence of G107A, G301A, G103A, G219T, T74C and T135C for amplification, described amplimer is selected from: for SEQ ID NO.43 and the SEQ ID NO.44 of G107A, SEQ ID NO.45 and SEQ ID NO.46 for G301A, SEQ ID NO.47 and SEQ ID NO.48 for G103A, SEQ ID NO.49 and SEQ ID NO.50 for G219T, SEQ ID NO.51 and SEQ ID NO.52 for T74C, SEQ ID NO.53 and SEQ ID NO.54 for T163C, for more than a pair of in the SEQ ID NO.55 of T135C and SEQ ID NO.56.
2. karyomit(e) 6q23 section polymorphic detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is selected from: for the sequence be comprised of SEQ ID NO.1 and SEQ ID NO.15 of G107A and the sequence be comprised of SEQ ID NO.2 and SEQ ID NO.16, for the sequence formed by SEQ ID NO.3 and SEQ ID NO.17 of G301A and the sequence formed by SEQ ID NO.4 and SEQ ID NO.18, for the sequence formed by SEQ ID NO.5 and SEQ ID NO.19 of G103A and the sequence formed by SEQ ID NO.6 and SEQ ID NO.20, for the sequence formed by SEQ ID NO.7 and SEQ ID NO.21 of G219T and the sequence formed by SEQ ID NO.8 and SEQ ID NO.22, for the sequence formed by SEQ ID NO.9 and SEQ ID NO.23 of T74C and the sequence formed by SEQ ID NO.10 and SEQ ID NO.24, for the sequence formed by SEQ ID NO.11 and SEQ ID NO.25 of T163C and the sequence formed by SEQ ID NO.12 and SEQ ID NO.26, for more than a pair of in the sequence formed by SEQ ID NO.13 and SEQ ID NO.27 of T135C and the sequence that formed by SEQ ID NO.14 and SEQ ID NO.28.
3. karyomit(e) 6q23 section polymorphic detection liquid-phase chip according to claim 1, is characterized in that, mainly includes:
A. specificity ASPE primer: for the sequence formed by SEQ ID NO.1 and SEQ ID NO.15 of G107A and the sequence formed by SEQ ID NO.2 and SEQ ID NO.16, for the sequence formed by SEQ ID NO.3 and SEQ ID NO.17 of G301A and the sequence formed by SEQ ID NO.4 and SEQ ID NO.18, for the sequence formed by SEQ ID NO.5 and SEQ ID NO.19 of G103A and the sequence formed by SEQ ID NO.6 and SEQ ID NO.20, for the sequence formed by SEQ ID NO.7 and SEQ ID NO.21 of G219T and the sequence formed by SEQ ID NO.8 and SEQ ID NO.22, for the sequence formed by SEQ ID NO.9 and SEQ ID NO.23 of T74C and the sequence formed by SEQ ID NO.10 and SEQ ID NO.24, for the sequence formed by SEQ ID NO.11 and SEQ ID NO.25 of T163C and the sequence formed by SEQ ID NO.12 and SEQ ID NO.26, with the sequence formed by SEQ ID NO.13 and SEQ ID NO.27 for T135C and the sequence formed by SEQ ID NO.14 and SEQ ID NO.28,
B. be coated with respectively the microballoon of special anti-tag sequence, described anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing, described anti-tag sequence is selected from the sequence in SEQ ID NO.29～SEQ ID NO.42, in the middle of described anti-tag sequence is connected with microballoon, also is provided with the spacerarm sequence;
C. described amplimer is: for the SEQ ID NO.43 of G107A and SEQ ID NO.44, for the SEQ ID NO.45 of G301A and SEQ ID NO.46, for the SEQ ID NO.47 of G103A and SEQ ID NO.48, for the SEQ ID NO.49 of G219T and SEQ ID NO.50, for the SEQ ID NO.51 of T74C and SEQ ID NO.52, for the SEQ ID NO.53 of T163C and SEQ ID NO.54 with for SEQ ID NO.55 and the SEQ ID NO.56 of T135C.
4. according to the described karyomit(e) 6q23 of claim 1-3 any one section polymorphic detection liquid-phase chip, it is characterized in that, described spacerarm sequence is 5-10 T.
5. the Auele Specific Primer for karyomit(e) 6q23 section polymorphic detection, it is characterized in that, described specific primer sequence is: for SEQ ID NO.15 and the SEQ ID NO.16 of G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, for more than a pair of in the SEQ ID NO.27 of T135C and SEQ ID NO.28.
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| CN101633963A (en) * | 2009-08-11 | 2010-01-27 | 广州益善生物技术有限公司 | Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof |
| CN101988897A (en) * | 2009-08-07 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Liquid phase chip detector based on quantum dot |
| CN102031286A (en) * | 2010-06-08 | 2011-04-27 | 广州益善生物技术有限公司 | Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer |
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| CN101988897A (en) * | 2009-08-07 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Liquid phase chip detector based on quantum dot |
| CN101633963A (en) * | 2009-08-11 | 2010-01-27 | 广州益善生物技术有限公司 | Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof |
| CN102031286A (en) * | 2010-06-08 | 2011-04-27 | 广州益善生物技术有限公司 | Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer |
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