CN102304567A - Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome - Google Patents
Polymorphic detection specific primers and liquid phase chip in 8 q 24 section of chromosome Download PDFInfo
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Abstract
The invention discloses specific primers and liquid phase chip for polymorphic detection in an 8 q 24 section of a chromosome. The liquid phase chip comprises specific primers consisting of tag sequences at the 5' end and specific primer sequences aiming at polymorphic loci of a target gene at the 3' end, microspheres and an amplification primer, wherein the specific primer sequences are selected from more than one pair of SEQ ID No. 23 and SEQ ID No. 24 for G83T, SEQ ID No. 25 and SEQ ID No. 26 for C140A, SEQ ID No. 27 and SEQ ID No. 28 for C160T, SEQ ID No. 29 and SEQ ID No. 30 for G99A, SEQ ID No. 31 and SEQ ID No. 32 for C202A, SEQ ID No. 33 and SEQ ID No. 34 for C296T, SEQ ID No. 35 and SEQ ID No. 36 for A125C, SEQ ID No. 37 and SEQ ID No. 38 for T119C, SEQ ID No. 39 and SEQ ID No. 40 for C149A, SEQ ID No. 41 and SEQ ID No. 42 for C193T and SEQ ID No. 43 and SEQ ID No. 44 for G201T; and the tag sequences are selected from SEQ ID No. 1 to SEQ ID No. 22. The prepared liquid phase chip for the polymorphic detection in the 8 q 24 section of the chromosome have a high signal noise ratio, and cross reaction is not formed between a designed probe and anti-tag sequences basically.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of karyomit(e) 8q24 zone detection Auele Specific Primer and the liquid-phase chip of relating to.
Background technology
Full genome association study finds that (single nucleotide polymorphisms, SNPs) genetic predisposition with kinds of tumors such as prostate cancer, colorectal carcinoma and mammary cancer is relevant for the single nucleotide polymorphism of karyomit(e) 8q24 section.
At present, the method that the single nucleotide polymorphism of karyomit(e) 8q24 section is detected, analyzes is a lot, as: direct sequencing, PCR-RFLP analytical method, traditional solid phase chip or the like, wherein the most frequently used is 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; Through a certain particular segment of pcr amplification; Use the digestion with restriction enzyme amplified production again; The segmental size of electrophoresis observation; This method is used to detect the transgenation that restriction enzyme site changes, and can directly judge genotype, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Once more, traditional solid phase chip can not satisfy the needs of practical application because of its poor repeatability, and the method for other PCR-based technology all exists the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
The karyomit(e) 8q24 block mutation site of target detect of the present invention, it is as shown in the table:
Summary of the invention
The karyomit(e) 8q24 section polymorphism that provides one of the object of the invention detects liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of 11 kinds of common pleomorphism site G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T of karyomit(e) 8q24 section.
A kind of karyomit(e) 8q24 section polymorphism detects liquid-phase chip, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively to 11 kinds of common pleomorphism sites of karyomit(e) 8q24 section; Every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence to the goal gene pleomorphism site of 3 ' end, and said specific primer sequence is selected from respectively: to SEQ ID NO.23 and the SEQ ID NO.24 of G83T; SEQ ID NO.25 and SEQ ID NO.26 to C140A; SEQ ID NO.27 and SEQ ID NO.28 to C160T; SEQ ID NO.29 and SEQ ID NO.30 to G99A; SEQID NO.31 and SEQ ID NO.32 to C202A; SEQ ID NO.33 and SEQ ID NO.34 to C296T; SEQID NO.35 and SEQID NO.36 to A125C; SEQ ID NO.37 and SEQ ID NO.38 to T119C; SEQ ID NO.39 and SEQ ID NO.40 to C149A; SEQ ID NO.41 and SEQ ID NO.42 to C193T; To more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44; Said tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.22;
B. be coated with the microballoon of special anti-tag sequence respectively; Said anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.45～SEQ ID NO.66; Also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. be used for increasing have G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T the primer of an above pleomorphism site target sequence.
Preferably, said amplimer is: to SEQ ID NO.67 and the SEQ ID NO.68 of G83T; SEQID NO.69 and SEQ ID NO.70 to C140A; SEQ ID NO.71 and SEQ ID NO.72 to C160T; SEQ ID NO.73 and SEQ ID NO.74 to G99A; SEQ ID NO.75 and SEQ ID NO.76 to C202A; SEQ ID NO.77 and SEQ ID NO.78 to C296T; SEQ ID NO.79 and SEQ ID NO.80 to A125C; SEQ ID NO.81 and SEQID NO.82 to T119C; SEQ ID NO.83 and SEQ ID NO.84 to C149A; SEQ ID NO.85 and SEQ IDNO.86 to C193T; To more than a pair of among the SEQ ID NO.87 of G201T and the SEQ ID NO.88.
Preferably, said ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.23 to G83T reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.24; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.25 to C140A reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.26; The sequence of being made up of SEQ ID NO.5 and SEQ IDNO.27 to C160T reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.28; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.29 to G99A reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.30; The sequence of being made up of SEQID NO.9 and SEQ ID NO.31 to C202A reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.32; The sequence of being made up of SEQ ID NO.11 and SEQ ID NO.33 to C296T reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.34; The sequence of being made up of SEQ ID NO.13 and SEQ ID NO.35 to A125C reaches the sequence of being made up of SEQ ID NO.14 and SEQ ID NO.36; The sequence of being made up of SEQ ID NO.15 and SEQ ID NO.37 to T119C reaches the sequence of being made up of SEQ ID NO.16 and SEQID NO.38; The sequence of being made up of SEQ ID NO.17 and SEQ ID NO.39 to C149A reaches the sequence of being made up of SEQ IDNO.18 and SEQ ID NO.40; The sequence of being made up of SEQ ID NO.19 and SEQ ID NO.41 to C193T reaches the sequence of being made up of SEQ ID NO.20 and SEQ ID NO.42; More than a pair of in the sequence of forming to the sequence of forming by SEQ ID NO.21 and SEQ ID NO.43 of G201T and by SEQ ID NO.22 and SEQ ID NO.44.
Another object of the present invention provides a kind of specific primer sequence that karyomit(e) 8q24 section polymorphism detects that is used for.
The technical scheme that realizes above-mentioned purpose is following:
A kind of specific primer sequence that is used for the detection of karyomit(e) 8q24 section polymorphism, it is selected from: to SEQ IDNO.23 and the SEQ ID NO.24 of G83T; SEQ ID NO.25 and SEQ ID NO.26 to C140A; SEQ ID NO.27 and SEQ ID NO.28 to C160T; SEQ ID NO.29 and SEQ ID NO.30 to G99A; SEQ ID NO.31 and SEQ ID NO.32 to C202A; SEQ ID NO.33 and SEQ ID NO.34 to C296T; SEQ ID NO.35 and SEQID NO.36 to A125C; SEQ ID NO.37 and SEQ ID NO.38 to T119C; SEQ ID NO.39 and SEQ IDNO.40 to C149A; SEQ ID NO.41 and SEQ ID NO.42 to C193T; To more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44.
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, meets the practical application needs especially.Because in very a large amount of Auele Specific Primers, through test in a large number, reaction is verified, obtains the liquid-phase chip system of optimum combination.Prepared karyomit(e) 8q24 section polymorphism detects liquid-phase chip and has extraordinary signal-noise ratio; And there is not cross reaction between institute's designed probe and the anti-tag sequence basically; Choosing of tag sequence label, anti-tag sequence label and combining of tag sequence label and concrete ASPE primer; Can avoid cross reaction, realize the parallel detection in a plurality of SNP site.
2. the present invention has chosen optimum combination through the design experiences of contriver's long-term accumulation and a large amount of experimental implementation from numerous Auele Specific Primers.The genotype of various types is accurately distinguished in the mutational site that designed ASPE primers Auele Specific Primer of the present invention can sensitive recognition objective specifically detects; In same reaction system, between the different Auele Specific Primers, do not have cross reaction basically 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 site mutation situation, also the polymorphism situation in a plurality of mutational sites of parallel detection simultaneously detects the effect unanimity.
3. detection method step of the present invention is simple; 11 kinds of pleomorphism sites detect and can accomplish 11 amplifications that contain the target sequence in SNP site through a step multiplex PCR; Many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided; Thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1 karyomit(e) 8q24 section polymorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
To wild-type and the mutant of 11 kinds of common genotype G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T of karyomit(e) 8q24 section, design specific primers sequence respectively.The ASPE primer is made up of " Tag sequence+specific primer sequence ".The ASPE primer sequence is as shown in the table:
The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 karyomit(e) 8q24 section
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or the special primer fragment (shown in above-mentioned table 1) of wild-type.All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon that encapsulates of anti-tag sequence
According to institute's designed ASPE Auele Specific Primer fragment; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and ASPE Auele Specific Primer fragment possibly form, corresponding anti-tag sequence is as shown in table 2 on 22 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
22 kinds of microballoons selecting encapsulate the anti-tag sequence on microballoon available from U.S. Luminex company.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, promptly 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.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Said spacerarm is to be used for anti-tag and microsphere surface is spaced apart or anti-tag placed the sequence of hydrophilic environments.Through 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 like (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 encapsulates is following:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/m1).EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/LTris (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 pleomorphism site
11 kinds of common pleomorphism site G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T to karyomit(e) 8q24 section; Design of amplification primers amplifies 11 target sequences that contain pleomorphism site respectively 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 the stock solution of 100pmol/mL respectively with 10mmol/LTris Buffer.
Embodiment 2 utilization karyomit(e) 8q24 zone detection liquid-phase chips are to the detection of sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250m1) 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, obtain DNA to be detected about DNA extraction.
Two, the pcr amplification of testing sample
Design 11 pairs of primers; Multiplex PCR one step amplifies 11 kinds of common genotype G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T totally 11 the target sequence that contains karyomit(e) 8q24 section respectively; The product size is respectively 273bp, 342bp, 307bp, 351bp, 362bp, 471bp, 283bp, 353bp, 440bp, 385bp and 432bp, and primer sequence (SEQ ID NO.67-88) is seen shown in the above-mentioned table 3.
At first prepare the multiple PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ ID NO.67-88 respectively mixes and is the multiple PCR primer working fluid in the 1.5ml Eppendorf tube.The multi-PRC reaction system is following:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are subsequent use.
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 directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize above-mentioned designed ASPE primers to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: get site to be detected corresponding wild type and mutant ASPE primer stock solution 10ul respectively in the 1.5ml Eppendorf tube; Add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, (every kind of microballoon concentration is 2.5 * 10 to the corresponding 22 kinds of microballoons of every group selection
5Individual/ml);
2. the microballoon of getting every kind of numbering of 1ul respectively is in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in >=centrifugal the 2-5min of 3000g;
11. microballoon is resuspended in 1 * Tm hybridization buffer of 75ul, adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Six, the result detects and data analysis
The reaction after product is through Luminex serial analysis instrument detecting.Detected result is shown in table 4, table 5 and table 6.Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect 20 increments karyomit(e) 8q24 section pleomorphism site originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): 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 karyomit(e) 8q24 sector type detected result and the sequencing result rate of coincideing originally and reaches 100%.Can detect karyomit(e) 8q24 section polymorphic position vertex type exactly it is thus clear that karyomit(e) 8q24 section polymorphism provided by the present invention detects liquid-phase chip, 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 dyeing body 8q24 block mutation ratio (%)
Table 7 sample dyeing body 8q24 block mutation ratio (%)
Table 8 sample dyeing body 8q24 block mutation type analysis result
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | 296CT | 296CT |
| 3 | Wild-type | Wild-type |
| 4 | 149AA | 149AA |
| 5 | Wild-type | Wild-type |
| 6 | 99GA、125CC | 99GA、125CC |
| 7 | Wild-type | Wild-type |
| 8 | 83TT | 83TT |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | 202AA | 202AA |
| 13 | Wild-type | Wild-type |
| 14 | 160TT | 160TT |
| 15 | 125AC | 125AC |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | 201TT | 201TT |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of karyomit(e) 8q24 section pleomorphism site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with karyomit(e) 8q24 section C160T and C202A site mutation is example; Respectively to the wild-type of C160T and C202A 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 IDNO.1-SEQ ID NO.22; Accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that encapsulates on microballoon is selected from SEQ IDNO.45-SEQ ID NO.66.Specifically design shown in following table (table 9).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
The design of table 9 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 21-40 is detected, detected result is following:
Table 10 pattern detection result and Polymorphism Analysis
Table 11 pattern detection result and Polymorphism Analysis
| Sequence number NO. | The C202A-w detected value | The C202A-m detected value | The C202A judged result |
| Group4 | Group5 | Group6 | Group4 | Group5 | Group6 | Group4 | Group5 | Group6 | |
| Negative control | 25 | 16 | 20 | 24 | 22 | 16 | - | - | - |
| 21 | 3453 | 3994 | 3157 | 129 | 63 | 90 | 3％ | 1％ | 2％ |
| 22 | 3171 | 3750 | 2776 | 188 | 85 | 164 | 5％ | 2％ | 5％ |
| 23 | 3481 | 4193 | 3495 | 188 | 121 | 232 | 5％ | 2％ | 6％ |
| 24 | 2743 | 3616 | 2662 | 75 | 66 | 203 | 2％ | 1％ | 7％ |
| 25 | 3107 | 3669 | 2771 | 202 | 80 | 150 | 5％ | 2％ | 5％ |
| 26 | 2823 | 3591 | 2918 | 266 | 127 | 200 | 8％ | 3％ | 6％ |
| 27 | 2727 | 3362 | 2710 | 129 | 90 | 224 | 4％ | 2％ | 7％ |
| 28 | 3387 | 4065 | 3446 | 244 | 109 | 213 | 6％ | 2％ | 5％ |
| 29 | 171 | 120 | 133 | 2485 | 3367 | 2561 | 94％ | 97％ | 96％ |
| 30 | 2787 | 3408 | 2476 | 112 | 112 | 164 | 3％ | 3％ | 6％ |
| 31 | 3447 | 4078 | 3121 | 157 | 95 | 150 | 4％ | 2％ | 4％ |
| 32 | 2882 | 3430 | 2813 | 233 | 112 | 174 | 7％ | 3％ | 5％ |
| 33 | 2499 | 3364 | 2423 | 194 | 114 | 115 | 6％ | 3％ | 4％ |
| 34 | 2600 | 3291 | 2301 | 243 | 121 | 164 | 8％ | 3％ | 6％ |
| 35 | 2752 | 3680 | 2995 | 157 | 86 | 188 | 5％ | 2％ | 5％ |
| 36 | 2337 | 3297 | 2425 | 231 | 128 | 261 | 8％ | 3％ | 9％ |
| 37 | 2549 | 3327 | 2487 | 108 | 91 | 194 | 3％ | 2％ | 7％ |
| 38 | 2370 | 3301 | 2564 | 125 | 122 | 249 | 4％ | 3％ | 8％ |
| 39 | 2464 | 3162 | 2371 | 209 | 125 | 125 | 7％ | 3％ | 4％ |
| 40 | 3296 | 3825 | 2933 | 167 | 110 | 238 | 4％ | 2％ | 7％ |
Other is to 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 for use, 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 4 karyomit(e) 8q24 section polymorphism detection specificity primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detecting liquid-phase chip with karyomit(e) 8q24 section A125C and C193T site mutation is example; Respectively to the wild-type of A125C and C193T and the specific primer sequence of mutant design ASPE primer 3 ' end; Comprise preferred specific primer sequence and two alternative specific primer sequences in the embodiment of the invention 1, as shown in table 11.Where,
inside the base as a polymorphism.
Table 12 specific primer sequence
Detecting liquid-phase chip with karyomit(e) 8q24 section A125C and C193T site mutation is example; Respectively to the wild-type of A125C and C193T and the specific primer sequence of mutant design ASPE primer 3 ' end; The tag sequence of ASPE primer 5 ' end then is fixed as the best effect sequence among the embodiment 1; And select corresponding with it anti-tag sequence for use, specifically design shown in following table (table 13).It is said like embodiment 1 and embodiment 2 that synthetic, the anti-tag sequence of ASPE primer encapsulates microballoon, amplimer, detection method.
Two of the design of table 13 liquid-phase chip preparation
Two, sample detection
Adopt the liquid-phase chip of above-mentioned designing and preparing, by embodiment 2 said testing processes and method sample 41-60 is detected, detected result is following:
Table 14 pattern detection result and Polymorphism Analysis
Table 15 pattern detection result and Polymorphism Analysis
Other is to the liquid-phase chip in different mutational sites, and the ASPE primer uses different specific primer 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 specific primer sequence and tag sequence for use, and effect better (signal to noise ratio is better) is referring to present embodiment test group 7 and test group 10.Other different specific primer sequences and the collocation of tag sequence, with coming to the same thing of embodiment 2 and present embodiment, concrete data are omitted.
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Claims (6)
1. a karyomit(e) 8q24 section polymorphism detects liquid-phase chip, it is characterized in that, mainly includes:
A. wild-type and the mutant specificity ASPE primer that designs respectively to 11 kinds of common pleomorphism sites of karyomit(e) 8q24 section; Every kind of ASPE primer is made up of the tag sequence of 5 ' end and the specific primer sequence to the goal gene pleomorphism site of 3 ' end, and said specific primer sequence is selected from respectively: to SEQ ID NO.23 and the SEQ ID NO.24 of G83T; SEQ ID NO.25 and SEQID NO.26 to C140A; SEQ ID NO.27 and SEQ ID NO.28 to C160T; SEQ ID NO.29 and SEQ ID NO.30 to G99A; SEQ ID NO.31 and SEQ ID NO.32 to C202A; SEQ ID NO.33 and SEQ ID NO.34 to C296T; SEQ ID NO.35 and SEQ ID NO.36 to A125C; SEQ ID NO.37 and SEQ ID NO.38 to T119C; SEQ ID NO.39 and SEQ ID NO.40 to C149A; SEQ ID NO.41 and SEQ ID NO.42 to C193T; To more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44; Said tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.22;
B. be coated with the microballoon of special anti-tag sequence respectively; Said anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.45～SEQ ID NO.66; Also be provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon, above-mentioned every kind of microballoon has the different colours coding;
C. be used for increasing have G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T the primer of an above pleomorphism site target sequence.
2. karyomit(e) 8q24 section polymorphism according to claim 1 detects liquid-phase chip; It is characterized in that said amplimer is selected from: to SEQ ID NO.67 and the SEQ ID NO.68 of G83T; SEQ ID NO.69 and SEQ ID NO.70 to C140A; SEQ ID NO.71 and SEQ ID NO.72 to C160T; SEQ ID NO.73 and SEQ ID NO.74 to G99A; SEQ ID NO.75 and SEQ ID NO.76 to C202A; SEQ ID NO.77 and SEQ ID NO.78 to C296T; SEQ ID NO.79 and SEQ ID NO.80 to A125C; SEQ ID NO.81 and SEQ ID NO.82 to T119C; SEQ ID NO.83 and SEQ ID NO.84 to C149A; SEQ ID NO.85 and SEQ ID NO.86 to C193T; To more than a pair of among the SEQ ID NO.87 of G201T and the SEQ ID NO.88.
3. karyomit(e) 8q24 section polymorphism according to claim 1 detects liquid-phase chip; It is characterized in that said ASPE primer is selected from: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.23 to G83T reaches the sequence of being made up of SEQ ID NO.2 and SEQ ID NO.24; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.25 to C140A reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.26; The sequence of being made up of SEQ ID NO.5 and SEQ ID NO.27 to C160T reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.28; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.29 to G99A reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.30; The sequence of being made up of SEQ ID NO.9 and SEQ IDNO.31 to C202A reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.32; The sequence of being made up of SEQ IDNO.11 and SEQ ID NO.33 to C296T reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.34; The sequence of being made up of SEQ ID NO.13 and SEQ ID NO.35 to A125C reaches the sequence of being made up of SEQ ID NO.14 and SEQ ID NO.36; The sequence of being made up of SEQ ID NO.15 and SEQ ID NO.37 to T119C reaches the sequence of being made up of SEQ ID NO.16 and SEQ IDNO.38; The sequence of being made up of SEQ ID NO.17 and SEQ ID NO.39 to C149A reaches the sequence of being made up of SEQ IDNO.18 and SEQ ID NO.40; The sequence of being made up of SEQ ID NO.19 and SEQ ID NO.41 to C193T reaches the sequence of being made up of SEQ ID NO.20 and SEQ ID NO.42; More than a pair of in the sequence of forming to the sequence of forming by SEQ ID NO.21 and SEQ IDNO.43 of G201T and by SEQ ID NO.22 and SEQ ID NO.44.
4. karyomit(e) 8q24 section polymorphism according to claim 1 detects liquid-phase chip, it is characterized in that,
A. said specificity ASPE primer is: the sequence of being made up of SEQ ID NO.1 and SEQ ID NO.23 to G83T reaches the sequence of being made up of SEQID NO.2 and SEQ ID NO.24; The sequence of being made up of SEQ ID NO.3 and SEQ ID NO.25 to C140A reaches the sequence of being made up of SEQ ID NO.4 and SEQ ID NO.26; The sequence of being made up of SEQ ID NO.5 and SEQ IDNO.27 to C160T reaches the sequence of being made up of SEQ ID NO.6 and SEQ ID NO.28; The sequence of being made up of SEQ ID NO.7 and SEQ ID NO.29 to G99A reaches the sequence of being made up of SEQ ID NO.8 and SEQ ID NO.30; The sequence of being made up of SEQ ID NO.9 and SEQ ID NO.31 to C202A reaches the sequence of being made up of SEQ ID NO.10 and SEQ ID NO.32; The sequence of being made up of SEQ ID NO.11 and SEQ ID NO.33 to C296T reaches the sequence of being made up of SEQ ID NO.12 and SEQ ID NO.34; The sequence of being made up of SEQ ID NO.13 and SEQ ID NO.35 to A125C reaches the sequence of being made up of SEQ ID NO.14 and SEQ ID NO.36; The sequence of being made up of SEQ ID NO.15 and SEQ ID NO.37 to T119C reaches the sequence of being made up of SEQ ID NO.16 and SEQ ID NO.38; The sequence of being made up of SEQID NO.17 and SEQ ID NO.39 to C149A reaches the sequence of being made up of SEQ ID NO.18 and SEQ ID NO.40; The sequence of being made up of SEQ ID NO.19 and SEQ ID NO.41 to C193T reaches the sequence of being made up of SEQ ID NO.20 and SEQ ID NO.42; Reach the sequence of forming by SEQ ID NO.22 and SEQ ID NO.44 with the sequence of forming by SEQ ID NO.21 and SEQ ID NO.43 to G201T;
B. be coated with the microballoon of special anti-tag sequence respectively; Said anti-tag sequence can be correspondingly with A in selected tag sequence complementary pairing; Said anti-tag sequence is selected from the sequence among SEQ ID NO.45～SEQ ID NO.66, also is provided with the spacerarm sequence in the middle of said anti-tag sequence is connected with microballoon;
C. said amplimer is: to SEQ ID NO.67 and the SEQ ID NO.68 of G83T; SEQ ID NO.69 and SEQID NO.70 to C140A; SEQ ID NO.71 and SEQID NO.72 to C160T; SEQ ID NO.73 and SEQ ID NO.74 to G99A; SEQ ID NO.75 and SEQ ID NO.76 to C202A; SEQ ID NO.77 and SEQ ID NO.78 to C296T; SEQ ID NO.79 and SEQ ID NO.80 to A125C; SEQ ID NO.81 and SEQ ID NO.82 to T119C; SEQ ID NO.83 and SEQ ID NO.84 to C149A; SEQ ID NO.85 and SEQ ID NO.86 to C193T; With SEQ ID NO.87 and SEQ ID NO.88 to G201T.
5. detect liquid-phase chip according to each described karyomit(e) 8q24 section polymorphism of claim 1-4, it is characterized in that said spacerarm sequence is 5-10 T.
6. one kind is used for the Auele Specific Primer that karyomit(e) 8q24 section polymorphism detects; It is characterized in that said specific primer sequence is: to SEQ ID NO.23 and the SEQ ID NO.24 of G83T; SEQ ID NO.25 and SEQ ID NO.26 to C140A; SEQ ID NO.27 and SEQ ID NO.28 to C160T; SEQ ID NO.29 and SEQ ID NO.30 to G99A; SEQ ID NO.31 and SEQ ID NO.32 to C202A; SEQ ID NO.33 and SEQ ID NO.34 to C296T; SEQ ID NO.35 and SEQ ID NO.36 to A125C; SEQ ID NO.37 and SEQ ID NO.38 to T119C; SEQ ID NO.39 and SEQ ID NO.40 to C149A; SEQ ID NO.41 and SEQ ID NO.42 to C193T; To more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44.
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| CN102191336A (en) * | 2011-06-09 | 2011-09-21 | 广州益善生物技术有限公司 | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip |
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| CN101641451A (en) * | 2006-10-27 | 2010-02-03 | 解码遗传学私营有限责任公司 | Cancer susceptibility variants on the chr8q24.21 |
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| CN102191336A (en) * | 2011-06-09 | 2011-09-21 | 广州益善生物技术有限公司 | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip |
| CN102191336B (en) * | 2011-06-09 | 2013-04-24 | 广州益善生物技术有限公司 | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip |
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