CN102304567B - 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
Genome-wide association study finds that (single nucleotide polymorphisms, SNPs) is relevant with the genetic predisposition of the kinds of tumors such as prostate cancer, colorectal carcinoma and mammary cancer 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 etc., 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, 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, 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
One of purpose of the present invention provides karyomit(e) 8q24 section polymorphic detection liquid-phase chip, and this liquid-phase chip can be used for detecting wild-type and the mutant of 11 kinds of common polymorphisms 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 polymorphic detection liquid-phase chip mainly includes:
A. the wild-type and the mutant specificity ASPE primer that design respectively for 11 kinds of common polymorphisms sites of karyomit(e) 8q24 section, 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, and described specific primer sequence is selected from respectively: for SEQ ID NO.23 and the SEQ ID NO.24 of 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, SEQID NO.31 and SEQ ID NO.32 for C202A, SEQ ID NO.33 and SEQ ID NO.34 for C296T, SEQID NO.35 and SEQID 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, for more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.22;
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 among SEQ ID NO.45～SEQ ID NO.66, 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. be used for amplification have G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T the primer of an above pleomorphism site target sequence.
Preferably, described amplimer is: for SEQ ID NO.67 and the SEQ ID NO.68 of G83T, SEQID NO.69 and SEQ ID NO.70 for C140A, SEQ ID NO.71 and SEQ ID NO.72 for C160T, SEQ ID NO.73 and SEQ ID NO.74 for G99A, SEQ ID NO.75 and SEQ ID NO.76 for C202A, SEQ ID NO.77 and SEQ ID NO.78 for C296T, SEQ ID NO.79 and SEQ ID NO.80 for A125C, SEQ ID NO.81 and SEQID NO.82 for T119C, SEQ ID NO.83 and SEQ ID NO.84 for C149A, SEQ ID NO.85 and SEQ IDNO.86 for C193T, for more than a pair of among the SEQ ID NO.87 of G201T and the SEQ ID NO.88.
Preferably, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.23 for G83T reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.25 for C140A reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.26, the sequence that is comprised of SEQ ID NO.5 and SEQ IDNO.27 for C160T reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.29 for G99A reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.30, the sequence that is comprised of SEQID NO.9 and SEQ ID NO.31 for C202A reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.33 for C296T reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.34, the sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.35 for A125C reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.36, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.37 for T119C reaches the sequence that is comprised of SEQ ID NO.16 and SEQID NO.38, the sequence that is comprised of SEQ ID NO.17 and SEQ ID NO.39 for C149A reaches the sequence that is comprised of SEQ IDNO.18 and SEQ ID NO.40, the sequence that is comprised of SEQ ID NO.19 and SEQ ID NO.41 for C193T reaches the sequence that is comprised of SEQ ID NO.20 and SEQ ID NO.42, more than a pair of in the sequence that forms for the sequence that is formed 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 for karyomit(e) 8q24 section polymorphic detection.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of specific primer sequence for karyomit(e) 8q24 section polymorphic detection, it is selected from: for SEQ IDNO.23 and the SEQ ID NO.24 of 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 SEQID NO.36 for A125C, SEQ ID NO.37 and SEQ ID NO.38 for T119C, SEQ ID NO.39 and SEQ IDNO.40 for C149A, SEQ ID NO.41 and SEQ ID NO.42 for C193T, for 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, and realistic especially application needs.Because 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) 8q24 section polymorphic detection liquid-phase chip 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 in a plurality of SNP site.
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, 11 kinds of pleomorphism sites detect and can finish 11 amplifications that contain the target sequence in SNP site by a step multiplex PCR, many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided, thereby can 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.
Embodiment
Embodiment 1 karyomit(e) 8q24 section polymorphic detection liquid-phase chip mainly includes:
One, ASPE primer
Wild-type and mutant for 11 kinds of common genotype G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T of karyomit(e) 8q24 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) 8q24 section
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 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 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/m1) 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/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 polymorphisms site G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, C193T and G201T for karyomit(e) 8q24 section, design of amplification primers amplifies respectively 11 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/LTris Buffer.
Embodiment 2 uses karyomit(e) 8q24 zone detection liquid-phase chip to the detection of sample
The prescription of described various solution is as follows:
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 about DNA extraction, obtain DNA to be detected.
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 respectively karyomit(e) 8q24 section, 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: respectively get respectively the primer stock solution 100ul of SEQ ID NO.67-88 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 in site 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 is 2.5 * 10 to the corresponding 22 kinds of microballoons of every group selection
5Individual/ml);
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 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 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.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 karyomit(e) 8q24 sector type detected result and the sequencing result rate of coincideing originally and reaches 100%.As seen karyomit(e) 8q24 section polymorphic detection liquid-phase chip provided by the present invention can detect karyomit(e) 8q24 section polymorphic position vertex type exactly, 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
Detect liquid-phase chip as example take karyomit(e) 8q24 section C160T and C202A site mutation, respectively for 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 is coated on the microballoon is selected from SEQ IDNO.45-SEQ ID NO.66.Specific design is shown in following table (table 9).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 9 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 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 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 4 karyomit(e) 8q24 section polymorphic detection specific primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take karyomit(e) 8q24 section A125C and C193T site mutation, respectively for 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.Wherein,
Interior base is pleomorphism site.
Table 12 specific primer sequence
Detect liquid-phase chip as example take karyomit(e) 8q24 section A125C and C193T site mutation, respectively for 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 with it corresponding anti-tag sequence, specific design is shown in following table (table 13).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 13 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 14 pattern detection result and Polymorphism Analysis
Table 15 pattern detection result and Polymorphism Analysis
Other is for 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, 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 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 (5)
1. a karyomit(e) 8q24 section polymorphic detection liquid-phase chip is characterized in that, mainly includes:
A. the wild-type and the mutant specificity ASPE primer that design respectively for 11 kinds of common polymorphisms sites of karyomit(e) 8q24 section, 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, and described specific primer sequence is selected from respectively: for SEQ ID NO.23 and the SEQ ID NO.24 of 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, for more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44; Described tag sequence is selected from the sequence among SEQ ID NO.1～SEQ ID NO.22;
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 among SEQ ID NO.45～SEQ ID NO.66, 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. be used for amplification and have G83T, C140A, C160T, G99A, C202A, C296T, A125C, T119C, C149A, the primer of an above pleomorphism site target sequence among C193T and the G201T, described amplimer is selected from: for SEQ ID NO.67 and the SEQ ID NO.68 of G83T, SEQ ID NO.69 and SEQ ID NO.70 for C140A, SEQ ID NO.71 and SEQ ID NO.72 for C160T, SEQ ID NO.73 and SEQ ID NO.74 for G99A, SEQ ID NO.75 and SEQ ID NO.76 for C202A, SEQ ID NO.77 and SEQ ID NO.78 for C296T, SEQ ID NO.79 and SEQ ID NO.80 for A125C, SEQ ID NO.81 and SEQ ID NO.82 for T119C, SEQ ID NO.83 and SEQ ID NO.84 for C149A, SEQ ID NO.85 and SEQ ID NO.86 for C193T, for more than a pair of among the SEQ ID NO.87 of G201T and the SEQ ID NO.88.
2. karyomit(e) 8q24 section polymorphic detection liquid-phase chip according to claim 1, it is characterized in that described ASPE primer is selected from: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.23 for G83T reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.25 for C140A reaches the sequence that is comprised of SEQ ID NO.4 and SEQID NO.26, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.27 for C160T reaches the sequence that is comprised of SEQ IDNO.6 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.29 for G99A reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.30, the sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.31 for C202A reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.33 for C296T reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.34, the sequence that is comprised of SEQID NO.13 and SEQ ID NO.35 for A125C reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.36, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.37 for T119C reaches the sequence that is comprised of SEQ ID NO.16 and SEQ ID NO.38, the sequence that is comprised of SEQ ID NO.17 and SEQ ID NO.39 for C149A reaches the sequence that is comprised of SEQ ID NO.18 and SEQ IDNO.40, the sequence that is comprised of SEQ ID NO.19 and SEQ ID NO.41 for C193T reaches the sequence that is comprised of SEQ ID NO.20 and SEQ ID NO.42, more than a pair of in the sequence that forms for the sequence that is formed by SEQ ID NO.21 and SEQ ID NO.43 of G201T and by SEQ ID NO.22 and SEQ ID NO.44.
3. karyomit(e) 8q24 section polymorphic detection liquid-phase chip according to claim 1 is characterized in that,
A. described specificity ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.23 for G83T reaches the sequence that is comprised of SEQID NO.2 and SEQ ID NO.24, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.25 for C140A reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.26, the sequence that is comprised of SEQ ID NO.5 and SEQ IDNO.27 for C160T reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.28, the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.29 for G99A reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.30, the sequence that is comprised of SEQ ID NO.9 and SEQ ID NO.31 for C202A reaches the sequence that is comprised of SEQ ID NO.10 and SEQ ID NO.32, the sequence that is comprised of SEQ ID NO.11 and SEQ ID NO.33 for C296T reaches the sequence that is comprised of SEQ ID NO.12 and SEQ ID NO.34, the sequence that is comprised of SEQ ID NO.13 and SEQ ID NO.35 for A125C reaches the sequence that is comprised of SEQ ID NO.14 and SEQ ID NO.36, the sequence that is comprised of SEQ ID NO.15 and SEQ ID NO.37 for T119C reaches the sequence that is comprised of SEQ ID NO.16 and SEQ ID NO.38, the sequence that is comprised of SEQ ID NO.17 and SEQ ID NO.39 for C149A reaches the sequence that is comprised of SEQ ID NO.18 and SEQ ID NO.40, the sequence that is comprised of SEQ ID NO.19 and SEQ ID NO.41 for C193T reaches the sequence that is comprised of SEQ ID NO.20 and SEQ ID NO.42, reach the sequence that is formed by SEQ ID NO.22 and SEQ ID NO.44 with the sequence that is formed by SEQ ID NO.21 and SEQ ID NO.43 for G201T;
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 among SEQ ID NO.45～SEQ ID NO.66, also is provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon;
C. described amplimer is: for SEQ ID NO.67 and the SEQ ID NO.68 of G83T, SEQ ID NO.69 and SEQ ID NO.70 for C140A, SEQ ID NO.71 and SEQ ID NO.72 for C160T, SEQ ID NO.73 and SEQ ID NO.74 for G99A, SEQ ID NO.75 and SEQ ID NO.76 for C202A, SEQ ID NO.77 and SEQ ID NO.78 for C296T, SEQ ID NO.79 and SEQ ID NO.80 for A125C, SEQ ID NO.81 and SEQ ID NO.82 for T119C, SEQ ID NO.83 and SEQ ID NO.84 for C149A, SEQ ID NO.85 and SEQ ID NO.86 for C193T, with SEQ ID NO.87 and the SEQ ID NO.88 for G201T.
4. each described karyomit(e) 8q24 section polymorphic detection liquid-phase chip is characterized in that described spacerarm sequence is 5-10 T according to claim 1-3.
5. Auele Specific Primer that is used for karyomit(e) 8q24 section polymorphic detection, it is characterized in that described specific primer sequence is: for SEQ ID NO.23 and the SEQ ID NO.24 of 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, for more than a pair of among the SEQ ID NO.43 of G201T and the SEQ ID NO.44.
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| WO2010141362A1 (en) * | 2009-05-31 | 2010-12-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for diagnosing the occurrence or likelihood of occurrence of testicular germ cell cancer |
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| WO2010141362A1 (en) * | 2009-05-31 | 2010-12-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for diagnosing the occurrence or likelihood of occurrence of testicular germ cell cancer |
Non-Patent Citations (4)
| Title |
|---|
| Associations between variants of the 8q24 chromosome and nine smoking related cancer sites;Park SL et al;《Cancer Epidemiol Biomarkers Prev》;20081130;全文 * |
| Park SL et al.Associations between variants of the 8q24 chromosome and nine smoking related cancer sites.《Cancer Epidemiol Biomarkers Prev》.2008,全文. |
| 人类染色体8q24.1带36个单拷贝的克隆与筛选;黎伶俐等;《中国现代医学杂志》;19941231;全文 * |
| 黎伶俐等.人类染色体8q24.1带36个单拷贝的克隆与筛选.《中国现代医学杂志》.1994,全文. |
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