CN103374601A - Specific primers and liquid-phase chip for chromosome 5p15 region SNP detection - Google Patents
Specific primers and liquid-phase chip for chromosome 5p15 region SNP detection Download PDFInfo
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Abstract
The invention provides a liquid-phase chip for chromosome 5p15 region SNP detection. The liquid-phase chip mainly comprises: ASPE primers composed of tag sequences of 5' end and sequences of specific primers for SNP sites of target genes of 3' end, wherein the sequences of the specific primers are SEQ ID No.9 and SEQ ID No.10 for C101T site, SEQ ID No.11 and SEQ ID No.12 for C107T site, SEQ ID No.13 and SEQ ID No.14 for C105A site, and/or SEQ ID No.15 and SEQ ID No.16 for C150T site; microballoons coated by anti-tag sequences; and amplimers. The liquid-phase chip for chromosome 5p15 region SNP detection and a sequencing method reach 100% agreement in detection results, and single or parallel detection of wide type and SNP type of four SNP sites can be realized.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of karyomit(e) 5p15 section SNP detection specificity primer and the liquid-phase chip of relating to.
Background technology
The TERT-CLPTM1L gene region is positioned at karyomit(e) 5p15.33 section, and it is relevant with the occurrence risk of kinds of tumors that the TERT-CLPTM1L gene pleiomorphism has been in the news.There are two known genes in this zone, telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) and harelip cross-film 1 sample albumen (cleft lip and palate transmembrane 1 like, CLPTM1L) gene.Biochemistry, physiological function and oncological pathology meaning in view of these two genes, this gene region is very fully nasopharyngeal carcinoma susceptibility genes candidate locus of biological evidences, and the present invention will be to target SNP site C101T, C107T, the C105A of nasopharyngeal carcinoma genetic predisposition with C150T separates and parallel detection.
The karyomit(e) 5p15 block mutation site of target detect of the present invention, it is as shown in the table:
| Order is special | The content of karyomit(e) 5p15 section site mutation | Write a Chinese character in simplified form |
| 1 | C → T sudden change occurs in the 101st Nucleotide of SEQ ID NO.41 | C101T |
| 2 | C → T sudden change occurs in the 107th Nucleotide of SEQ ID NO.42 | C107T |
| 3 | C → A sudden change occurs in the 105th Nucleotide of SEQ ID NO.43 | C105A |
| 4 | C → T sudden change occurs in the 150th Nucleotide of SEQ ID NO.44 | C150T |
SEQ ID NO.41 C101T
CTACGCACAGACGGCACTCACGAGGTGTGGAGGGCCGAGCTGCCACGTCTATGCATAGTGGGCAGAAAA
CAAGGTCTGCTATCCAGACAACTTCAGAGTC
ATCATGGTGTGAAGCAGCTTTCTGGCTGGTAAGTTTA
TCAAGAGTCTACGACAACTTTGGAGAGCAAAGCTCTTCTATTTATTAATCAGTGTAACTGCTGCCCACG
GGGTCAAGTCAGTGAGAGCACTGGAGCACCCTG。
SEQ ID NO.42 C107T
GCTGCTCCTTGTCGCCTGAGGAGTAGAGGAAGTGCTTGGTCTCGGCGTACACCGGGGGACAAGGCGTGT
CCCAGGGACGTGGTGGCCGCGATGTGGATGGGGGGCC
GCGTGGTGCTGGCGGCCCACGGATGGGTGGG
AGTGGCGCGTGCCAGAGAGCGCACCCTCCAAAGAGGTGGCTTCTTCGGCGGGTCTGGCAGGTGACACCA
CACAGAAACCACGG。
SEQ ID NO.43 C105A
CTTCACCCGCACACCAGGGCCCGACGTCCATACTACAGCCCCATGGAAAAACCTCGCATTCCACCTGTT
TACGGTTACATGAGTTCTTCTTCCTCTTTAAAAGT
TCTTTTTTGAGACAAGGTCTCGCTGTCACCAGG
CTGAAAGTGTAGGGGTGCAATCACAGCTCACTGCATCCTCAACCTCCTGAGCTCAAGTGACCTCCCGCC
TCAGTCTCCCGAGTAGCTGGGACTACAGGTGGGCGTGCCACCATGCCTGGCTAACTTTGAAAACTGTTG
TAGAGATGGGGTTTTGTCACATTGCC。
SEQ ID NO.44 C150T
CCCAAGTTTAGGCAAGACAGGAAAAACCACCACCTGCAAATTATCTTTTCCCTCAAATGGATAAACAGG
CGCAGGGTGCGGTGAAAGCCGTCATTCCGTTCAGCAGCAGCCACGCCGCTGAGACGGAGCAACGGCCGA
GCATACGCAGC
GCACTCACCACCGCTGGTACAGGTAGACCAGAAACACCACGTCGTCCCGGAAGCAGG
CCAGCCGGTGAGACGTGGGCATGGTGATGATGAAGGCAAAGACGTCATCAATGAAGGTGTTGAAAGCCT
GCAGGGCCAGACGGGAGGAGGGTGAAC。
At present, the common method that detects karyomit(e) 5p15.33 section SNP sudden change mainly contains the mass-spectrometric technique based on Sequenom, TaqMan method and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) classifying method, wherein, the characteristics of Sequenom mass-spectrometric technique are to use ddNTP to substitute dNTP, making probe only extend a base in the SNP site namely stops, by probe in conjunction with different ddNTP, thereby has different molecular weight to distinguish genotype, have the high characteristics of accuracy, but, this technology detects a kind of sudden change at every turn only, has the limitation that detects flux; Easy and simple to handle, the advantages such as the result quick, quantification that the TaqMan method has, still, this technology exists sample easily to pollute, and cross reaction easily occurs, the shortcoming that false positive rate is high; And the PCR-RFLP method is based on the change of the restriction enzyme enzyme recognition site that transgenation causes, as losing or produce novel site in the site, by a certain specific fragment of pcr amplification, use again the digestion with restriction enzyme amplified production, the size of electrophoresis observation fragment, this method can directly be judged genotype for detection of the transgenation that restriction enzyme site changes, but this method can not be used for not producing the detection in Gene Mutation of new restriction enzyme site.Again, more than these methods all exist the limitation that detects flux, can only detect a kind of mutation type at every turn, can not satisfy the needs of practical application.
Summary of the invention
One of purpose of the present invention provides karyomit(e) 5p15 section SNP and detects liquid-phase chip, and this liquid-phase chip can be used for separately or wild-type and the mutant of parallel detection karyomit(e) 5p15 section four kinds of common genotype C101T, C107T, C105A and C150T.
Realize that the above-mentioned purpose technical scheme is as follows:
A kind of karyomit(e) 5p15 section SNP detects liquid-phase chip, includes:
(A). the wild-type that designs respectively for the different SNP of karyomit(e) 5p15 section site and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for goal gene SNP site, described specific primer sequence is: for SEQ ID NO.9 and the SEQ IDNO.10 in C101T site, SEQ ID NO.11 and SEQ ID NO.12 for the C107T site, for SEQID NO.13 and the SEQ ID NO.14 in C105A site, and/or for SEQ ID NO.15 and the SEQ IDNO.16 in C150T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B). be coated with the microballoon anti-tag sequence, that have the different colours coding, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.17～SEQID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). being used for amplifying need to primer that detect, that have the target sequence in corresponding SNP site.
Therein among embodiment, described amplimer is: for SEQ ID NO.25 and the SEQ ID NO.26 in C101T site, SEQ ID NO.27 and SEQ ID NO.28 for the C107T site, for SEQ ID NO.29 and the SEQ ID NO.30 in C105A site, and/or for SEQ ID NO.31 and the SEQID NO.32 in C150T site.
Therein among embodiment, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 for the C101T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for the C107T site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.12, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.13 for the C105A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, and/or reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.16 for the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.15 in C150T site.
Another object of the present invention provides the Auele Specific Primer for karyomit(e) 5p15 section polymorphic detection.
Realize that the above-mentioned purpose technical scheme is as follows:
The Auele Specific Primer that is used for karyomit(e) 5p15 section polymorphic detection, described specific primer sequence is: for SEQ ID NO.9 and the SEQ ID NO.10 in C101T site, SEQ ID NO.11 and SEQ ID NO.12 for the C107T site, for SEQ ID NO.13 and the SEQ ID NO.14 in C105A site, and/or for SEQ ID NO.15 and the SEQ ID NO.16 in C150T site.
Major advantage of the present invention is:
1. the karyomit(e) 5p15 section SNP provided by the present invention detected result that detects liquid-phase chip and the identical rate of sequencing be up to 100%, and the needed time well below sequencing technologies commonly used, realistic especially application needs.Prepared karyomit(e) 5p15 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 of a plurality of pleomorphism sites.
2. 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. 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.
4. in addition, detection method step of the present invention is simple, 4 kinds of SNP sites are detected and can be finished 4 amplifications that contain the target sequence in SNP site by a step 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.
Embodiment
Embodiment 1
The described karyomit(e) 5p15 of present embodiment section polymorphic detection liquid-phase chip mainly is comprised of following:
One, ASPE primer
Wild-type and mutant for karyomit(e) 5p15 section four kinds of common genotype C101T, C107T, C105A and C150T 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) 5p15 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 8 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 2 microballoon numbering and the microballoon
8 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.Synthetic anti-tag sequence is made into the stock solution of 100nmol/ml with sterilization ddH2O.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 50ul0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/ml) of 10ul.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid that adds 2.5ul in the microballoon suspension, constant-temperature incubation 30 minutes adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/L Tris (pH8.0)] of 100ul, and among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For karyomit(e) 5p15 section four kinds of common genotype C101T, C107T, C105A and C150T, design of amplification primers amplifies respectively four target sequences that contain pleomorphism site to (seeing Table 3).
Table 3 amplifies the primer of the target sequence with pleomorphism site
All primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
Embodiment 2 uses embodiment 1 described karyomit(e) 5p15 section polymorphic detection liquid-phase chip to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MTris-HCl，pH8. | Sigma T3038 | 0.2M | 50ml |
| 5M NaCl | 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 four pairs of primers, one step of multiplex PCR amplifies the amplified production that contains respectively karyomit(e) 5p15 section four kinds of common genotype C101T, C107T, C105A and C150T, the product size is respectively 240bp, 221bp, 302bp and 303bp, and primer sequence (SEQ ID NO.25-32) 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.25-32 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 0.5ulExo-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 (table 1) of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare the ASPE primer working fluid that mixes: get respectively the corresponding wild-type of gene to be detected and mutant ASPE primer stock solution 10ul in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:
Response procedures is: 96 ℃ of 2min; 94 ℃ of 30s, 54 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization
According to the design the ASPE primer, the corresponding microballoon of every group selection (as described in Example 1), microballoon concentration is 2.5 * 10
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 ddH2O of 25ul;
6. get the ASPE reaction solution of 5-25ul in corresponding hole, complement to 50ul with ddH2O;
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 synchronous detection 20 increments 4 pleomorphism sites of karyomit(e) 5p15 section 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) 5p15 sector type detected result and the sequencing result rate of coincideing originally and reaches 100%.As seen karyomit(e) 5p15 section polymorphic detection liquid-phase chip provided by the present invention can detect karyomit(e) 5p15 section polymorphic position vertex type exactly, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 sample dyeing body 5p15 block mutation ratio (%)
Table 6 sample dyeing body 5p15 block mutation type analysis result
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of karyomit(e) 5p15 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) 5p15 section C107T, C150T, C101T and C105A site mutation, respectively for the wild-type of C107T, C150T, C101T and C105A and the specific primer sequence of mutant design ASPE primer 3 ' end, the tag sequence of ASPE primer 5 ' end then is selected from SEQ ID NO.1-SEQID NO.8, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.17-SEQ ID NO.24.Specific design is shown in following table (table 7).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 7 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 8 pattern detection result and Polymorphism Analysis
Table 9 pattern detection result and Polymorphism Analysis
Table 10 pattern detection result and Polymorphism Analysis
Table 11 pattern detection result and Polymorphism Analysis
From above-described embodiment as seen, the Tag sequence is combined into the ASPE primer applicable to different Auele Specific Primers, is used for described detection system.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, test group 5, test group 7 and test group 11.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) 5p15 section polymorphic detection specific primer sequences
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Pleomorphism site take karyomit(e) 5p15 section C101T and C105A detects liquid-phase chip as example, take the forward or backwards complementary sequence of this place, mutational site target sequence as template, respectively for the wild-type of C101T and C105A and the specific primer sequence of mutant design ASPE primer 3 ' end, comprise preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the invention 1, as shown in table 12.Wherein,
Interior base is pleomorphism site.
Table 12 specific primer sequence
Pleomorphism site take karyomit(e) 5p15 section C101T and C105A detects liquid-phase chip as example, select different specific primer sequences for C101T and C105A, 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
By present embodiment as seen, when the ASPE primer was selected among the embodiment 1 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better) was referring to present embodiment test group 13 and test group 16.Other derives from different specific primer sequences and the collocation of tag sequence of the forward or backwards complementary sequence of place, target detect site sequence, with coming to the same thing of embodiment 2 and present embodiment, namely still be that embodiment 1 is better from different tag sequence arranging effects with the specific primer sequence described in 2, concrete data are omitted.
Other is for multiple specific primer sequence and the collocation of tag sequence in different SNP sites, and with coming to the same thing of embodiment 2 and present embodiment, namely embodiment 1 selected Auele Specific Primer has better signal to noise ratio, and it is also better to detect effect, and concrete data are omitted.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. a karyomit(e) 5p15 section SNP detects liquid-phase chip, it is characterized in that, including: (A). the wild-type that designs respectively for the different SNP of karyomit(e) 5p15 section site and the ASPE primer of mutant: every kind of ASPE primer is comprised of the tag sequence of 5 ' end and 3 ' the end specific primer sequence for goal gene SNP site, described specific primer sequence is: for SEQ ID NO.9 and the SEQ IDNO.10 in C101T site, SEQ ID NO.11 and SEQ ID NO.12 for the C107T site, for SEQID NO.13 and the SEQ ID NO.14 in C105A site, and/or for SEQ ID NO.15 and the SEQ IDNO.16 in C150T site; Described tag sequence is selected from SEQ ID NO.1～SEQ ID NO.8;
(B). be coated with the microballoon anti-tag sequence, that have the different colours coding, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.17～SEQID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). being used for amplifying need to primer that detect, that have the target sequence in corresponding SNP site.
2. karyomit(e) 5p15 section SNP according to claim 1 detects liquid-phase chip, it is characterized in that, described amplimer is: for SEQ ID NO.25 and the SEQ ID NO.26 in C101T site, SEQ ID NO.27 and SEQ ID NO.28 for the C107T site, for SEQ ID NO.29 and the SEQID NO.30 in C105A site, and/or for SEQ ID NO.31 and the SEQ ID NO.32 in C150T site.
3. karyomit(e) 5p15 section SNP according to claim 1 detects liquid-phase chip, it is characterized in that, described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 for the C101T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for the C107T site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.12, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.13 for the C105A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, and/or reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.16 for the sequence that is comprised of SEQ ID NO.7 and SEQ IDNO.15 in C150T site.
4. karyomit(e) 5p15 section SNP according to claim 1 detects liquid-phase chip, it is characterized in that, (A). described ASPE primer is: the sequence that is comprised of SEQ ID NO.1 and SEQ ID NO.9 for the C101T site reaches the sequence that is comprised of SEQ ID NO.2 and SEQ ID NO.10, the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.11 for the C107T site reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.12, the sequence that is comprised of SEQ ID NO.5 and SEQ ID NO.13 for the C105A site reaches the sequence that is comprised of SEQ ID NO.6 and SEQ ID NO.14, and reaches the sequence that is comprised of SEQ ID NO.8 and SEQ ID NO.16 for the sequence that is comprised of SEQ ID NO.7 and SEQ ID NO.15 in C150T site; (B). be coated with the microballoon anti-tag sequence, that have the different colours coding, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.17～SEQID NO.24, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing; (C). described amplimer is: for SEQ ID NO.25 and the SEQ ID NO.26 in C101T site, SEQ ID NO.27 and SEQ ID NO.28 for the C107T site, for SEQ ID NO.29 and the SEQ ID NO.30 in C105A site, and for SEQ ID NO.31 and the SEQ ID NO.32 in C150T site.
5. each described karyomit(e) 5p15 section SNP detects liquid-phase chip according to claim 1-4, it is characterized in that described spacerarm is 5-10 T.
6. the Auele Specific Primer that is used for karyomit(e) 5p15 section polymorphic detection, it is characterized in that, described specific primer sequence is: for SEQ ID NO.9 and the SEQ ID NO.10 in C101T site, SEQ ID NO.11 and SEQ ID NO.12 for the C107T site, for SEQ ID NO.13 and the SEQ IDNO.14 in C105A site, and/or for SEQ ID NO.15 and the SEQ ID NO.16 in C150T site.
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| WO2002059354A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
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