CN102191336B - MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip - Google Patents
MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip Download PDFInfo
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Abstract
The invention discloses an MYC gene single nucleotide polymorphism (SNP) detection liquid-phase chip. The liquid-phase chip mainly comprises ASPE primers, a microsphere and an amplification primer, wherein each ASPE primer consists of a tag sequence of the 5' terminal and a specific primer for a target gene mutational site of the 3' terminal; the specific primers are one of SEQ ID NO. 1, SEQ ID NO.18 and SEQ ID NO.19 of a wild type for the G203T site, and one of SEQ ID NO.2, SEQ ID NO.20 and SEQ ID NO.21 of a mutant type for the G203T site; and the microsphere is coated by an anti-tag sequence. The coincidence rate between the detection result of the detection liquid-phase chip provided by the invention and a sequencing method is up to 100 percent; and parallel detection of the mild type and the mutant type of an SNP site is realized.
Description
Technical field
The present invention bends in biology field, relates to medical science and biotechnology, concrete a kind of MYC gene SNP detection specific primer and the liquid-phase chip of relating to.
Background technology
The MYC gene is one group of oncogene early finding, comprises C-MYC, N-MYC and L-MYC, is positioned respectively No. 8 karyomit(e)s, No. 2 karyomit(e)s and No. 1 karyomit(e).On the MYC gene structure by the 1st exon of coded protein and the 2nd, 3 exons of coded protein do not consist of.The MYC gene is bent in the oncogene of coding nucleoprotein, and 3 genes are all encoded a kind of nuclear dna relevant with cell cycle regulating in conjunction with albumen.MYC gene family and product thereof can promote cell proliferation, immortalization, dedifferente and conversion etc., is in critical role in the kinds of tumors forming process.
3 members in the MYC gene family form tumour and there are differences aspect the tumor type.At present research is thought, the expression of C-MYC and tumour occur and lapse to closely related, also play an important role in the cell death inducing process, the expression of N-MYC and the prognosis of tumour are judged meaningful, and the expression of L-MYC is different with susceptibility and the prognosis performance of tumour in different tumours.
The C-MYC gene mutation site of target detect of the present invention, it is as shown in the table:
| Sequence number | The content of MYC site mutation | Write a Chinese character in simplified form |
| 1 | G → T sudden change occurs in the 203rd Nucleotide of SEQ ID NO.17 | G203T |
At present, few to the report that the MYC gene SNP detects, analyzes, the method for its research mainly is direct sequencing and 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.
Summary of the invention
One of purpose of the present invention provides the MYC gene SNP detection liquid-phase chip.This liquid-phase chip can be used for detecting wild-type and the mutant of a kind of common genotype G203T of MYC gene.
A kind of MYC gene SNP detection liquid-phase chip includes:
(A). the wild-type that designs respectively for the G203T mutational 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 Auele Specific Primer for the goal gene mutational site, described Auele Specific Primer is: be selected among SEQ ID NO.1, SEQ ID NO.18 and the SEQ ID NO.19 one for the detection probes of wild-type, and be selected among SEQ ID NO.2, SEQ ID NO.20 and the SEQ ID NO.21 one for the detection probes of mutant; Described tag sequence is selected from any two among the SEQ ID NO.3-8;
(B). anti-tag sequence microballoon coated, that have the different colours coding is arranged, also be provided with the spacerarm sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected among SEQ ID NO.9～SEQ ID NO.14 two, 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 G203T SNP site.
Preferably, described wild-type detection probes is that SEQ ID NO.1, mutant detection probes are SEQ ID NO.2.
Preferably, described amplimer is SEQ ID NO.15～SEQ ID NO.16.
Preferably, described ASPE primer is: the sequence that is comprised of SEQ ID NO.3 and SEQ ID NO.1 reaches the sequence that is comprised of SEQ ID NO.4 and SEQ ID NO.2.
Another object of the present invention provides a kind of Auele Specific Primer for the detection of MYC gene SNP.
Concrete technical scheme is as follows:
A kind of Auele Specific Primer for the detection of MYC gene SNP includes SEQ ID NO.1 and SEQ ID NO.2.
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.Prepared MYC gene SNP 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 concrete ASPE primer of tag sequence label, anti-tag sequence label, and the combination of pcr amplification primer, cross reaction be can avoid, wild-type and the mutant parallel detection in SNP site realized.
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%.
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.
Embodiment
Embodiment 1MYC gene SNP detection liquid-phase chip mainly includes:
One, ASPE primer
Wild-type and mutant for the common genotype G203T of MYC gene 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:
Specific primer sequence in the table 1MYC Gene A SPE primer sequence
Tag sequence in the table 2MYC Gene A SPE primer sequence
| SEQ NO | The Tag sequence (5 '-3 ') of correspondence on the microballoon |
| 3 | CTTTATCAATACATACTACAATCA |
| 4 | CAATTCAAATCACAATAATCAATC |
| 5 | TACAAATCATCAATCACTTTAATC |
| 6 | CTTTTACAATACTTCAATACAATC |
| 7 | CTTTTCAAATCAATACTCAACTTT |
| 8 | TTACTACACAATATACTCATCAAT |
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 3 on 2 kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag sequence on table 3 microballoon numbering and the microballoon
| SEQ NO | The microballoon numbering | The anti-tag sequence (5 '-3 ') of correspondence on the microballoon |
| 9 | 28 | TGATTGTAGTATGTATTGATAAAG |
| 10 | 15 | GATTGATTATTGTGATTTGAATTG |
| 11 | 35 | GATTAAAGTGATTGATGATTTGTA |
| 12 | 25 | GATTGTATTGAAGTATTGTAAAAG |
| 13 | 51 | AAAGTTGAGTATTGATTTGAAAAG |
| 14 | 56 | ATTGATGAGTATATTGTGTAGTAA |
2 kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag sequence coated with microballoon on.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and the microballoon, namely add the spacerarm sequence of the preceding paragraph 5-10 T before each anti-tag sequence, the anti-tag sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is for being used for anti-tag and microsphere surface is spaced apart or anti-tag is placed the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between anti-tag sequence and microballoon, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are such as (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the process that microballoon is coated with is as follows:
Get respectively 5 * 10
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/ml) of 10ul.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.The EDC working fluid that adds 2.5ul in the microballoon suspension, constant-temperature incubation 30 minutes adds the EDC working fluid of 2.5ul again, and constant-temperature incubation is 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution [10mmol/LTris (pH8.0)] of 100ul, and among the lmmol/LEDTA, 2-8 ℃ keeps in Dark Place.
Three, amplify the primer of the target sequence that contains the mutational site
For the common genotype G203T of MYC gene, design of amplification primers amplifies the target sequence that contains the SNP site to (seeing Table 4).
Table 4 amplifies the primer of the target sequence with SNP site
| SEQ NO | The SNP site | Type | Amplimer (5 '-3 ') |
| 15 | G203T | G203T-Forward | CTTACTCTGTTGCCCTGGCTG |
| 16 | G203T-Reverse | GGTTCACTTCAACATTCCCCTCC |
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 applicable described MYC gene test liquid-phase chips are to the detection of sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5MNaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
Be stored in 4 ℃ after the filtration.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " methods involving about DNA extraction, obtain DNA to be detected.
Two, with the pcr amplification of test sample product
One step of design primer PCR amplifies the target sequence of the common genotype G203T that contains the MYC gene, and the product size is 468bp, and primer sequence (SEQ ID NO.15-16) 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.15-16 in the 1.5ml Eppendorf tube, mix and be the multiple PCR primer working fluid.The multi-PRC reaction system is as follows:
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min; 4 ℃ save backup.
Three, the enzyme of PCR product is cut processing
1. get the reacted product of 7.5ul PCR, add 1ul 10 * SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
2.37 ℃ hatch 15min, hatch 15min for 80 ℃, the enzyme that deactivation is unnecessary.The product that enzyme is cut after the processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare the ASPE primer working fluid that mixes: get respectively the corresponding wild-type of gene to be detected and mutant ASPE primer stock solution 10ul in the 1.5ml Eppendorf tube, the composition of ASPE primer is respectively:
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 2 kinds of coated 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 MYC gene SNP 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 MYC genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen MYC gene SNP detection liquid-phase chip provided by the present invention can detect MYC gene SNP site type exactly, and the result is reliable and stable.
One of table 4 pattern detection result (MFI)
Table 5 sample MYC transgenation ratio (%)
| Sample number | G203T |
| 1 | 1％ |
| 2 | 1％ |
| 3 | 1％ |
| 4 | 2％ |
| 5 | 1％ |
| 6 | 98％ |
| 7 | 50％ |
| 8 | 1％ |
| 9 | 1％ |
| 10 | 2％ |
| 11 | 1％ |
| 12 | 98％ |
| 13 | 1％ |
| 14 | 1％ |
| 15 | 1％ |
| 16 | 2％ |
| 17 | 2％ |
| 18 | 1％ |
| 19 | 1％ |
| 20 | 1％ |
Table 6 sample MYC gene mutation type analytical results
| Catalogue number(Cat.No.) | The liquid-phase chip detected result | Sequencing result |
| 1 | Wild-type | Wild-type |
| 2 | Wild-type | Wild-type |
| 3 | Wild-type | Wild-type |
| 4 | Wild-type | Wild-type |
| 5 | Wild-type | Wild-type |
| 6 | 203TT | 203TT |
| 7 | 203CT | 203CT |
| 8 | Wild-type | Wild-type |
| 9 | Wild-type | Wild-type |
| 10 | Wild-type | Wild-type |
| 11 | Wild-type | Wild-type |
| 12 | 203TT | 203TT |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | Wild-type | Wild-type |
| 18 | Wild-type | Wild-type |
| 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 MYC gene SNP site
One, the design (selection of Tag sequence and Anti-Tag sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take MYC gene G203T site mutation, respectively for the wild-type of G203T 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.3-SEQ IDNO.8, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing that is coated on the microballoon is selected from SEQ ID NO.9-SEQID NO.14.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 gene SNP analysis
Other is for the liquid-phase chip in G203T mutational site, 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 2 collocation of tag sequence and specific primer sequence, and effect better (signal to noise ratio is better) is referring to present embodiment test group 1.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 4MYC gene SNP detection specific primer sequence
One, the design (selection of wild-type and mutant specific primer sequence) of liquid-phase chip preparation
Detect liquid-phase chip as example take the SNP site of MYC gene G203T, take the forward or backwards complementary sequence of this place, mutational site target sequence as template, respectively for the wild-type of G203T 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 9.Wherein,
Interior base is pleomorphism site.
Table 9 specific primer sequence
Detect liquid-phase chip as example take the SNP site of MYC gene G203T, select different specific primer sequences for G203T, 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 10).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 10 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 11 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 4.Other derives from different specific primer sequences and the collocation of tag sequence of the forward or backwards complementary sequence of place, target detect site sequence, with coming to the same thing of embodiment 2 and present embodiment, namely still be that the specific primer sequence described in the embodiment 2 is better from different tag sequence arranging effects, concrete data are omitted.
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 (4)
1. a MYC gene SNP detection liquid-phase chip is characterized in that, includes:
(A). the wild-type that designs respectively for G203T SNP 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: be selected among SEQ ID NO.1, SEQ ID NO.18 and the SEQ ID NO.19 one for the detection probes of wild-type, and be selected among SEQ ID NO.2, SEQ ID NO.20 and the SEQ ID NO.21 one for the detection probes of mutant; Described tag sequence is selected from any two among the SEQ ID NO.3-8;
(B). by different anti-tag sequences two kinds of microballoons coated, that have the different colours coding, in the middle of being connected with microballoon, described anti-tag sequence also is provided with the spacerarm sequence respectively; Described anti-tag sequence is selected among SEQ ID NO.9～SEQ ID NO.14 one, and described anti-tag sequence can be correspondingly with (A) in selected tag sequence complementary pairing;
(C). need primer detection, that have the target sequence in G203T SNP site for amplifying, described amplimer is SEQ ID NO.15～SEQ ID NO.16.
2. MYC gene SNP detection liquid-phase chip according to claim 1 is characterized in that, described specific primer sequence is: be SEQ ID NO.1 and be SEQ ID NO.2 for the detection probes of mutant for the detection probes of wild-type.
3. each described MYC gene SNP detection liquid-phase chip is characterized in that according to claim 1-2, and described ASPE primer is: the primer that is comprised of SEQ ID NO.3 and SEQ ID NO.1 reaches the primer that is comprised of SEQ ID NO.4 and SEQ ID NO.2.
4. each described MYC gene SNP detection liquid-phase chip is characterized in that according to claim 1-2, and described spacerarm is 5-10 T.
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| 于雷等.c-myc 和k-ras 基因多态性与辐射诱发小鼠胸腺淋巴瘤的关联性分析.《吉林大学学报（医学版）》.2009,296-298. * |
| 徐保锋.脑胶质瘤病人遗传易感基因单核苷酸多态性的研究.《中国优秀博硕士学位论文全文数据库 (硕士) 医药卫生科技辑》.2005, |
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