CN103031367B - VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip - Google Patents
VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip Download PDFInfo
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Abstract
The invention discloses a VHL (Von Hippel Lindau) genetic mutation detection specific primer and a liquid phase chip. The liquid phase chip mainly comprises ASPE (Allele Specific Primer Extension) primers, microspheres coated by an anti-tag sequence, and amplimers, wherein the ASPE primers consist of tag sequences at a 5' end and specific primer sequences specific to target genetic mutation sites at a 3' end; and the specific primer sequences are: SEQ ID NO.13 and SEQ ID NO.14 specific to a T240A site; SEQ ID NO.15 and SEQ ID NO.16 specific to a T254C site; SEQ ID NO.17 and SEQ ID NO.18 specific to a T266A site; SEQ ID NO.19 and SEQ ID NO.20 specific to a T286T site; SEQ ID NO.21 and SEQ ID NO.22 specific to a G388C site; and/or SEQ ID NO.23 and SEQ ID NO.24 specific to a 444delT site. The matching rate between a detection result of the liquid phase chip provided by the invention and a sequencing method is up to 100 percent, and wild and mutant parallel detection of a plurality of mutant sites is realized.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a specific primer and a liquid chip for detecting VHL gene mutation.
Background
The VHL gene is known as Von Hippel-Lindau (VHL) disease and contains 14543bp, comprising 3 exons and 2 introns. VHL disease is a very rare familial autosomal dominant hereditary tumor disease in clinic, including central nervous system hemangioblastoma, visceral tumor and cyst, and the like, in 1993, Latif and the like locate VHL gene on chromosome 3p25-26 through linkage analysis, and successfully clone VHL gene for the first time. The VHL gene is an anti-cancer gene, and the inactivation of the gene can cause normal VHL protein synthesis disorder and has close relation with the generation and development of Clear Cell Renal Cell Carcinoma (CCRCC). Currently, research on the structure and function of the VHL gene and the regulatory pathway thereof is greatly advanced, and the inactivation mechanism of the VHL gene is clarified, mainly comprising gene mutation, heterozygous deletion and methylation. The invention mainly detects six common genotypes of the VHL gene, namely T240A, T254C, T266A, C286T, G388C and 444 delT.
At present, few methods are used for detecting and analyzing VHL gene mutation, and a direct sequencing method and a PCR-RFLP analysis method are mainly used, wherein the most common method is the PCR-RFLP analysis method. The PCR-RFLP method is based on the change of restriction enzyme recognition site caused by gene mutation, such as site loss or new site generation, a certain specific segment is amplified through PCR, the amplified product is cut by restriction enzyme, and the size of the segment is observed through electrophoresis. Thirdly, the methods have the limitation of detection flux, only one mutation type can be detected at a time, and the requirements of practical application cannot be met.
Disclosure of Invention
One of the purposes of the invention is to provide a VHL gene mutation detection liquid chip which can be used for detecting T240A, T254C, T266A, C286T, G388C and 444delT wild type and mutant of six common genotypes of a VHL gene independently or in parallel.
The technical scheme for realizing the purpose is as follows:
a liquid phase chip for detecting VHL gene mutation comprises:
(A) wild type and mutant ASPE primers designed for different mutation sites of the VHL gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: SEQ ID No.13 and SEQ ID No.14 for position T240A; SEQ ID No.15 and SEQ ID No.16 for position T254C; SEQ ID NO.17 and SEQ ID NO.18 for position T266A; SEQ ID No.19 and SEQ ID No.20 for position C286T; SEQ ID NO.21 and SEQ ID NO.22 for the G388C site; and/or SEQ ID No.23 and SEQ ID No.24 for the 444delT site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 12;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) primers for amplifying a target sequence to be detected having a corresponding mutation site.
Preferably, the amplification primers are SEQ ID No.37 and SEQ ID No.38 to positions T240A, T254C, T266A, C286T; and/or SEQ ID NO.39 and SEQ ID NO.40 for the G388C, 444delT site.
Preferably, the ASPE primer is a sequence which is aimed at the T240A site and consists of SEQ ID NO.1 and SEQ ID NO.13 and a sequence which consists of SEQ ID NO.2 and SEQ ID NO. 14; a sequence consisting of SEQ ID No.3 and SEQ ID No.15 and a sequence consisting of SEQ ID No.4 and SEQ ID No.16 directed to the T254C site; a sequence consisting of SEQ ID No.5 and SEQ ID No.17 and a sequence consisting of SEQ ID No.6 and SEQ ID No.18 for position T266A; the sequence consisting of SEQ ID NO.7 and SEQ ID NO.19 and the sequence consisting of SEQ ID NO.8 and SEQ ID NO.20 for position C286T; the sequence consisting of SEQ ID NO.9 and SEQ ID NO.21 and the sequence consisting of SEQ ID NO.10 and SEQ ID NO.22 for the G388C site; and/or a sequence consisting of SEQ ID NO.11 and SEQ ID NO.23 and a sequence consisting of SEQ ID NO.12 and SEQ ID NO.24 for the 444delT site.
It is another object of the present invention to provide specific primers for the detection of mutations in the VHL gene.
The technical scheme for realizing the purpose is as follows:
specific primers for the detection of mutations in the VHL gene are: SEQ ID No.13 and SEQ ID No.14 for position T240A; SEQ ID No.15 and SEQ ID No.16 for position T254C; SEQ ID NO.17 and SEQ ID NO.18 for position T266A; SEQ ID No.19 and SEQ ID No.20 for position C286T; SEQ ID NO.21 and SEQ ID NO.22 for position G388C; and/or SEQ ID NO.23 and SEQ ID NO.24 for the 444delT site.
The main advantages of the invention are:
1. the coincidence rate of the detection method provided by the invention and the sequencing method is up to 100%. And the time required by detection is far shorter than that of the common sequencing technology, and the method particularly meets the requirement of practical application. The prepared VHL gene mutation detection liquid phase chip has a very good signal-to-noise ratio, cross reaction does not exist between the designed probe and the anti-tag sequence basically, the tag sequence, the anti-tag sequence are selected, and the tag sequence is combined with a specific ASPE primer, so that the cross reaction can be avoided, and the parallel detection of a plurality of mutation sites can be realized.
2. The invention selects the optimal combination from a plurality of specific primers through the long-term accumulated design experience and a large amount of experimental operation of the inventor. The ASPE primer specific primer designed by the invention can sensitively and specifically identify the mutation site of target detection and accurately distinguish genotypes of various types; in the same reaction system, cross reaction basically does not exist among different specific primers and between the specific primers and a PCR amplification product for non-target detection, the detection specificity is good, and the cross reaction rate is lower than 3%; besides the mutation condition of a single site, the mutation conditions of a plurality of mutation sites can be simultaneously detected in parallel, and the detection effects are consistent.
3. The detection method has simple steps, can complete the amplification of 2 target sequences containing the mutation sites by one-step PCR for the detection of 6 mutation sites, and avoids a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like, thereby greatly improving the detection accuracy and embodying the accurate and qualitative and quantitative analysis characteristics.
4. The invention not only overcomes the defects of low sensitivity and poor repeatability of detection results of the traditional solid phase chip, but also improves the existing liquid phase chip technology, so that the prepared microspheres can be suitable for different detection items and have strong expansibility. The detected fluorescence signal value is greatly improved, so that the detection sensitivity is further improved, the signal-to-noise ratio is enhanced, and the detection result is more accurate and reliable.
Detailed Description
Example 1VHL gene mutation detection liquid phase chip, mainly including:
first, ASPE primer
Specific primer sequences are designed aiming at wild types and mutant types of six common genotypes of a VHL gene, namely T240A, T254C, T266A, C286T, G388C and 444 delT. The ASPE primer consists of a tag sequence and a specific primer sequence. The ASPE primer sequences are shown in the following table:
TABLE 1 ASPE primer sequences (tag sequence + specific primer sequence) of VHL genes
Each ASPE primer comprises two parts, the 5 'end is a specific tag sequence aiming at an anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant type or wild type specific primer segment (as shown in the table 1). All ASPE primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Two, anti-tag sequence coated microsphere
Selecting tag sequences according to the designed ASPE specific primer fragments, and reducing secondary structures possibly formed between anti-tag sequences of the microspheres and tag and the ASPE specific primer fragments to the maximum extent, wherein the numbers of the selected 12 microspheres and the corresponding anti-tag sequences on the microspheres are shown in Table 2:
TABLE 2 numbering of microspheres and corresponding anti-tag sequences on microspheres
Selected 12 microspheres were purchased from Luminex, usa and were coated with the anti-tag sequence. 5-10T spacer arm sequences are connected between the anti-tag sequences and the microspheres, namely a 5-10T spacer arm sequence is added in front of each anti-tag sequence, and the anti-tag sequences are synthesized by Shanghai Bioengineering technology service GmbH. The synthetic anti-tag sequence was treated with sterile ddH2O is prepared into a 100nmol/ml stock solution. The spacer arm is a sequence for spacing the anti-tag from the surface of the microsphere or placing the anti-tag in a hydrophilic environment. By using anti-tag sequenceAnd a spacer arm sequence with proper length is arranged between the microsphere and the probe, so that the steric hindrance can be reduced, and the efficiency of hybridization reaction and the specificity of the hybridization reaction are improved. Common spacer sequences include poly-dT, i.e., poly (dT), oligo-tetrapolyethylene glycol, and (CH2) n spacers (n.gtoreq.3), such as (CH2)12, (CH2)18, and the like. In addition, if a poly (dA) interference is present, poly (TTG) may also be used as a spacer. The spacer arm of the invention is preferably 5-10T, and the process of coating the microspheres is as follows:
respectively taking 5 × 106Each of the above numbered carboxylated microspheres (from Luminex) was suspended in 50. mu.l of 0.1mol/L MES solution (pH4.5) and 10. mu.l of synthetic anti-tag molecule (100nmol/ml) was added. 10ng/ml of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) working solution (available from Pierce Chemical Co.) 2.5ul of EDC working solution was added to the microsphere suspension, incubated at constant temperature for 30 minutes, 2.5ul of EDC working solution was added, incubated at constant temperature for 30 minutes, washed once with 0.02% Tween-20 and then 0.1% SDS after the completion of the reaction, and the washed microspheres coated with the anti-tag sequence were resuspended in 100ul of Tris-EDTA solution [10mmol/L Tris (pH8.0) ]]In 1mmol/L EDTA, storing at 2-8 deg.c in dark.
Thirdly, amplifying the primer of the target sequence containing the mutation site
Aiming at six common genotypes of the VHL gene, namely T240A, T254C, T266A, C286T, G388C and 444delT, an amplification primer pair is designed (see Table 3), and 2 target sequences containing 6 mutation sites are amplified.
TABLE 3 primers for amplifying target sequences with mutation sites
All primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was formulated with 10mmol/LTrisBuffer as a 100pmol/mL stock solution.
Example 2 detection of samples Using the VHL Gene mutation detection liquid chip described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH5.0) formulation (250 ml):
2 XTM hybridization buffer
| Reagent | Origin of origin | Final concentration | The dosage of each 250ml |
| 1MTris-HCl，pH8.0 | SigmaT3038 | 0.2M | 50ml |
| 5MNaCl | Sigma S5150 | 0.4M | 20ml |
| Triton X-100 | Sigma T8787 | 0.16％ | 0.4ml |
After filtration, the mixture was stored at 4 ℃.
The ExoSAP-IT kit was purchased from U.S. USB.
Biotin-labeled dCTP was purchased from Shanghai Biotechnology engineering services, Inc.
Firstly, DNA extraction of a sample:
the DNA to be detected is obtained by referring to the related method of DNA extraction in molecular cloning.
Second, PCR amplification of the sample to be tested
2 pairs of primers are designed, and 2 target sequences respectively containing six common genotypes of the VHL gene, namely T240A, T254C, T266A, C286T, G388C and 444delT are amplified by multiplex PCR in one step, wherein T240A, T254C, T266A and C286T are located in the same amplification product, and G388C and 444delT are located in the same amplification product. The sizes of the products were 367bp and 456bp, respectively, and the primer sequences (SEQ ID NO.37-40) were as shown in Table 3 above.
Firstly, preparing a multiplex PCR primer working solution: 100ul of primer stock solution of SEQ ID NO.37-40 is respectively taken and put in a 1.5ml microcentrifuge tube, and the multiple PCR primer working solution is obtained after uniform mixing. The multiplex PCR reaction system is as follows:
2 Xbuffer (containing Mg)2+) 25ul
dNTP (2.5 mmol/L each) 4ul
Taq enzyme (5U/ul) 0.2ul
6ul of multiplex PCR primer working solution (8.3 pmol/mL each)
Template DNA (10ng/ul) 2ul
ddH2O 12.8ul
Total 50ul
The PCR amplification procedure was: 3min at 95 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃; storing at 4 deg.C for use.
Thirdly, enzyme digestion treatment of PCR product
1. Taking 7.5ul of the product after PCR reaction, adding 1ul of 10 XSAP buffer solution, 1ul of SAP enzyme and 0.5ul of Exo-I enzyme;
incubate at 2.37 ℃ for 15min, incubate at 80 ℃ for 15min, inactivate excess enzyme. The product after enzyme digestion is directly used for the subsequent ASPE primer extension reaction.
Site-specific primer extension reaction (ASPE)
The primer extension reaction is carried out by using the ASPE primer designed above, and the biotin-labeled dCTP is incorporated during the reaction, so that the product after the reaction is labeled with a plurality of biotin.
Firstly, preparing mixed ASPE primer working solution: respectively taking 10ul of wild type and mutant ASPE primer stock solution corresponding to the gene to be detected, adding 10mmol/L Tris Buffer to supplement to 200ul, and uniformly mixing to obtain the ASPE mixed primer working solution. The system for the ASPE reaction is as follows:
10 Xbuffer 2ul
MgCl2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
1ul of mixed solution of dATP, dGTP and dTTP (100 umol/L each)
Tsp enzyme (5U/ul) 0.25ul
Mixed ASPE primer working solution (500 nmol/L each) 1ul
Enzyme-digested PCR amplification product 5ul
ddH2O 10ul
Total 20ul
The reaction procedure is as follows: 2min at 96 ℃; 30 cycles of 94 ℃ for 30s, 54 ℃ for 1min, 72 ℃ for 2 min; storing at 4 deg.C for use.
Fifthly, hybridization reaction
1. Based on the designed ASPE primers, 12 corresponding coated microspheres (as described in example 1) were selected per set, each at a concentration of 2.5X 105Per ml;
2. 1ul of microspheres with each number are respectively taken and put in a 1.5ml microcentrifuge tube;
3. centrifuging the microspheres at a speed of more than or equal to 10000g for 1-2 min;
4. discarding the supernatant, resuspending the microspheres in 100ul of 2 XTM hybridization buffer, and mixing by vortex;
5. 25ul of the microsphere suspension was placed in the corresponding well of a 96-well filter plate, and 25ul of ddH was added to the control well2O；
6. Taking 5-25ul ASPE reaction solution into corresponding holes, and using ddH2O is complemented to 50 ul;
7. wrapping a 96-well plate with tin foil paper to avoid light, and incubating and hybridizing at 95 ℃ for 60s and 37 ℃ for 15 min;
8. centrifuging the hybridized microspheres for 2-5min at a speed of more than or equal to 3000 g;
9. removing supernatant, and suspending the microspheres in 75ul of 1 XTM hybridization buffer;
10. centrifuging the microspheres at a speed of more than or equal to 3000g for 2-5 min;
11. resuspend the microspheres in 75ul of 1 XTM hybridization buffer, add 15ul of streptavidin-phycoerythrin (SA-PE) at 10 ug/ml;
incubate at 12.37 ℃ for 15min and detect on Luminex instruments.
Sixthly, result detection and data analysis
And detecting the product after reaction by a Luminex series analytical instrument. And taking the mutant fluorescence value (MFI) of more than 100 as a cut-off value, judging that the sample has the mutant type when the MFI value detected by the mutant is more than 100, and otherwise, judging that the sample is the corresponding wild type.
The method is used for detecting VHL gene mutation of a large number of samples, the detection of a sequencing method is compared with the result of a liquid chip, and the coincidence rate of the detection result of the method provided by the invention is calculated. The coincidence rate of the VHL genotype detection result of 20 samples detected by the method and the sequencing result reaches 100%. Therefore, the VHL gene mutation detection liquid-phase chip provided by the invention can accurately detect the mutation type of the VHL gene, and the result is stable and reliable.
TABLE 4 one of the sample test results (MFI)
TABLE 5 two sample test results (MFI)
TABLE 6 results of analysis of VHL Gene mutation types
| Sample number | Liquid phase chip detection result | Sequencing results |
| 1 | Wild type | Wild type |
| 2 | Wild type | Wild type |
| 3 | T240A mutation | T240A mutation |
| 4 | Wild type | Wild type |
| 5 | T266A mutation | T266A mutation |
| 6 | Wild type | Wild type |
| 7 | Wild type | Wild type |
| 8 | Wild type | Wild type |
| 9 | Wild type | Wild type |
| 10 | Wild type | Wild type |
| 11 | G388C mutation | G388C mutation |
| 12 | Wild type | Wild type |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | Wild type | Wild type |
| 16 | Wild type | Wild type |
| 17 | C286T mutation | C286T mutation |
| 18 | Wild type | Wild type |
| 19 | Wild type | Wild type |
| 20 | Wild type | Wild type |
EXAMPLE 3 detection of VHL Gene mutation site by liquid phase chip of different ASPE primers
Design of liquid phase chip preparation (tag sequence and Anti-tag sequence selection)
Taking VHL gene T240A and T254C site mutation detection liquid phase chip as an example, specific primer sequences at the 3 'end of ASPE primers are designed aiming at the wild type and the mutant type of T240A and T254C respectively, tag sequences at the 5' end of the ASPE primers are selected from SEQ ID NO.1-SEQ ID NO.12, and correspondingly, anti-tag sequences coated on microspheres and complementarily paired with the corresponding tag sequences are selected from SEQ ID NO.25-SEQ ID NO. 36. The specific design is shown in the following table (table 7). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 7 design of liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 21-40 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 8 sample test results and Gene mutation analysis
TABLE 9 sample test results and Gene mutation analysis
From the experimental results, the results of the ASPE primers are still stable and reliable by using different Tag sequences, but the results are better (the signal to noise ratio is better) when the Tag sequences in example 1 are selected and matched with the specific primer sequences in the ASPE primers, see test group 1 and test group 5 in this example. Other different tag sequences are matched with the specific primer sequences, the results are the same as those of the example 2 and the example, and specific data are omitted.
In other liquid phase chips aiming at different mutation sites, different Tag sequences are applied to the ASPE primers, the result is still stable and reliable, and when the Tag sequences in the embodiment 1 are selected to be matched with the specific primer sequences, the effect is better, and specific data are omitted.
Example 4 selection of primer sequences specific for detection of VHL Gene mutations
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking a liquid phase chip for detecting mutation sites of VHL genes T266A and G388C as an example, taking a forward or reverse complementary sequence of a target sequence where the mutation sites are located as templates, specific primer sequences at the 3' end of an ASPE primer are designed aiming at a wild type and a mutant type of T266A and G388C respectively, and the specific primer sequences comprise a preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment 1 of the invention, as shown in Table 10. Wherein,the internal base is a mutation site.
TABLE 10 specific primer sequences
Taking the liquid phase chip for detecting mutation sites of T266A and G388C of VHL genes as an example, different specific primer sequences are selected for T266A and G388C, and the tag sequence at the 5' end of the ASPE primer is fixed as the best effect sequence in example 1, and the anti-tag sequence corresponding to the optimal effect sequence is selected, and the specific design is shown in the following table (Table 11). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 11 design two for liquid phase chip preparation
Second, sample detection
The liquid phase chip prepared by the design is adopted to detect the samples 41-60 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 12 sample test results and Gene mutation analysis
TABLE 13 sample test results and Gene mutation analysis
In this example, the specific primer sequence in example 1 is selected as the ASPE primer to match with the tag sequence, which is better (better signal to noise ratio), see test group 7 and test group 10 in this example. Other different specific primer sequences derived from the forward or reverse complementary sequence of the target detection site are matched with the tag sequence, which is the same as the results of the embodiment 2 and the embodiment, i.e., the specific primer sequence described in the embodiment 1 is still better matched with different tag sequences, and specific data are omitted.
Other specific primer sequences aiming at the same mutation site or different mutation sites are matched with the tag sequence, and the result is the same as that of the embodiment 2 and the embodiment, namely the specific primer selected in the embodiment 1 has better signal to noise ratio and better detection effect, and specific data are omitted.
The above is a detailed description of possible embodiments of the present invention, but the embodiments are not intended to limit the scope of the present invention, and all equivalent implementations or modifications that do not depart from the technical spirit of the present invention are intended to be included in the scope of the present invention.
Claims (4)
1. A VHL gene mutation detection liquid chip is characterized by comprising:
(A) wild type and mutant ASPE primers designed for different mutation sites of the VHL gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequence is as follows: SEQ ID No.13 and SEQ ID No.14 for position T240A; SEQ ID No.15 and SEQ ID No.16 for position T254C; SEQ ID NO.17 and SEQ ID NO.18 for position T266A; SEQ ID No.19 and SEQ ID No.20 for position C286T; SEQ ID NO.21 and SEQ ID NO.22 for the G388C site; and/or SEQ ID No.23 and SEQ ID No.24 for the 444delT site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 12;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) a primer for amplifying a target sequence to be detected, which has a corresponding mutation site; the amplification primers are as follows: SEQ ID NO.37 and SEQ ID NO.38 for the T240A, T254C, T266A, C286T sites; and/or SEQ ID NO.39 and SEQ ID NO.40 for the G388C, 444delT site.
2. The liquid phase chip for detecting VHL gene mutation according to claim 1, wherein said ASPE primers are: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.13 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.14 for the T240A site; a sequence consisting of SEQ ID No.3 and SEQ ID No.15 and a sequence consisting of SEQ ID No.4 and SEQ ID No.16 for position T254C; a sequence consisting of SEQ ID NO.5 and SEQ ID NO.17 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.18 for the T266A site; a sequence consisting of SEQ ID NO.7 and SEQ ID NO.19 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.20 for position C286T; the sequence consisting of SEQ ID NO.9 and SEQ ID NO.21 and the sequence consisting of SEQ ID NO.10 and SEQ ID NO.22 for the G388C site; and/or a sequence consisting of SEQ ID NO.11 and SEQ ID NO.23 and a sequence consisting of SEQ ID NO.12 and SEQ ID NO.24 for the 444delT site.
3. The liquid phase chip for VHL gene mutation detection according to claim 1, wherein said chip is a chip for detecting VHL gene mutation,
(A) the ASPE primers are as follows: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.13 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.14 for the T240A site; a sequence consisting of SEQ ID No.3 and SEQ ID No.15 and a sequence consisting of SEQ ID No.4 and SEQ ID No.16 directed to the T254C site; a sequence consisting of SEQ ID No.5 and SEQ ID No.17 and a sequence consisting of SEQ ID No.6 and SEQ ID No.18 for position T266A; the sequence consisting of SEQ ID NO.7 and SEQ ID NO.19 and the sequence consisting of SEQ ID NO.8 and SEQ ID NO.20 for position C286T; the sequence consisting of SEQ ID NO.9 and SEQ ID NO.21 and the sequence consisting of SEQ ID NO.10 and SEQ ID NO.22 for the G388C site; and a sequence consisting of SEQ ID NO.11 and SEQ ID NO.23 and a sequence consisting of SEQ ID NO.12 and SEQ ID NO.24 for the 444delT site;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) the amplification primers are as follows: SEQ ID NO.37 and SEQ ID NO.38 for positions T240A, T254C, T266A, C286T; and SEQ ID NO.39 and SEQ ID NO.40 for the G388C, 444delT site.
4. The liquid phase chip for detecting VHL gene mutation according to any of claims 1 to 3, wherein said spacer arm has 5 to 10T's.
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