CN103849940A - Specific primer and liquid phase chip for detecting BARD1 gene mutation - Google Patents
Specific primer and liquid phase chip for detecting BARD1 gene mutation Download PDFInfo
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Abstract
The invention discloses a liquid phase chip and a specific primer for detecting BARD1 gene mutation. The liquid phase chip mainly comprises: ASPE primers, microspheres coated by different anti-tag sequences, and an amplification primer, wherein each ASPE primer is composed of a tag sequence at a 5' end and specific primer sequences at a 3' end and aiming at target gene mutation sites, and the specific primer sequences are as follows: SEQ ID NO.11 and SEQ ID NO.12 aiming at a T6883G site, SEQ ID NO.13 and SEQ ID NO.14 aiming at an A43635G site, SEQ ID NO.15 and SEQ ID NO.16 aiming at a G1670C site, SEQ ID NO.17 and SEQ ID NO.18 aiming at a G25542A site, and/or SEQ ID NO.19 and SEQ ID NO.20 aiming at a G1134C site. The coincidence rate of the detection result of the detection liquid phase chip disclosed by the invention with that of a sequencing method reaches as high as 100%, and parallel detection of a wild type and a mutant of a plurality of mutation sites is achieved.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a BARD1 gene mutation detection specific primer and a liquid chip.
Background
BRCA 1-associated Loop Domain 1 (BRCA 1 associated RING domain, BARD 1) is a protein that binds to BRCA1 protein and is designated BARD1 because it has a similar loop finger domain as BRCA 1. The BARD1 gene is located in the long arm of chromosome 2q34-35, and encodes a 777 amino acid residue-containing protein. The main functional domain of the protein has a ring finger functional domain, three ankyrin repetitive sequences and two carboxyl terminal functional domains. The repair of damaged DNA by the BARD1 protein can be prevented by mutation of the BARD1 gene, and the growth and division of cells can be out of control due to the accumulation of DNA defects, so that tumors are formed. In addition, the study finds that the BARD1 gene plays a certain role in the occurrence and development of breast cancer.
At present, the mutation detection method of the BARD1 gene mainly comprises the following steps: matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), fluorescence quantitative PCR technology and PCR-RFLP, wherein the matrix assisted laser desorption ionization time-of-flight mass spectrometry is soft ionization technology, has strong and mature functions in the detection of protein and other biomacromolecules, but in the field of nucleic acid detection, the detection is limited to a certain extent due to the specificity of nucleic acid molecules, the fluorescence quantitative PCR technology has the characteristics of high sensitivity, strong specificity and high automation degree, but also has the defects of easy sample pollution and high false positive rate, and can only detect one mutation type at each time, the PCR-RFLP method is based on the change of restriction endonuclease recognition sites caused by gene mutation, such as site loss or generation of new sites, a certain specific segment is amplified by PCR, and the amplified product is cut by restriction endonuclease, the size of the fragment is observed by electrophoresis, and the method is used for detecting the gene mutation with changed enzyme cutting sites and can directly judge the genotype, but the method cannot be used for detecting the gene mutation without generating new enzyme cutting sites.
Disclosure of Invention
One of the purposes of the invention is to provide a liquid phase chip for detecting BARD1 gene mutation, which can be used for detecting wild type and mutant type of five common genotypes of BARD1 gene T6883G, A43635G, G1670C, G25542A and G1134C separately or in parallel.
The technical scheme for achieving the purpose is as follows.
A liquid phase chip for detecting BARD1 gene mutation comprises:
(A) primer pairs of wild type and mutant ASPE designed for different mutation sites of BARD1 gene: 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.11 and SEQ ID NO.12 for the T6883G site, SEQ ID NO.13 and SEQ ID NO.14 for the A43635G site, SEQ ID NO.15 and SEQ ID NO.16 for the G1670C site, SEQ ID NO.17 and SEQ ID NO.18 for the G25542A site, and/or SEQ ID NO.19 and SEQ ID NO.20 for the G1134C site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 10;
(B) microspheres coated by different anti-tag sequences and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequences and the microspheres; the anti-tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30, 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.
In one embodiment, the amplification primers are: SEQ ID NO.31 and SEQ ID NO.32 for the T6883G site, SEQ ID NO.33 and SEQ ID NO.34 for the A43635G site, SEQ ID NO.35 and SEQ ID NO.36 for the G1670C site, SEQ ID NO.37 and SEQ ID NO.38 for the G25542A site, and/or SEQ ID NO.39 and SEQ ID NO.40 for the G1134C site.
In one embodiment, the ASPE primer pair is a sequence consisting of SEQ ID No.1 and SEQ ID No.11 and a sequence consisting of SEQ ID No.2 and SEQ ID No.12 for site T6883G, a sequence consisting of SEQ ID No.3 and SEQ ID No.13 and a sequence consisting of SEQ ID No.4 and SEQ ID No.14 for site a43635G, a sequence consisting of SEQ ID No.5 and SEQ ID No.15 and a sequence consisting of SEQ ID No.6 and SEQ ID No.16 for site G1670C, a sequence consisting of SEQ ID No.7 and SEQ ID No.17 and a sequence consisting of SEQ ID No.8 and SEQ ID No.18 for site G25542A, and/or a sequence consisting of SEQ ID No.9 and SEQ ID No.19 and a sequence consisting of SEQ ID No.10 and SEQ ID No.20 for site G1134C.
Another objective of the invention is to provide specific primers for the detection of the mutation of the BARD1 gene.
The technical scheme for achieving the purpose is as follows.
Specific primers for BARD1 gene mutation detection are SEQ ID NO.11 and SEQ ID NO.12 aiming at the T6883G site, SEQ ID NO.13 and SEQ ID NO.14 aiming at the A43635G site, SEQ ID NO.15 and SEQ ID NO.16 aiming at the G1670C site, SEQ ID NO.17 and SEQ ID NO.18 aiming at the G25542A site, and/or SEQ ID NO.19 and SEQ ID NO.20 aiming at the G1134C site.
The main advantages of the invention are:
1. the coincidence rate of the detection result of the liquid phase chip for detecting the BARD1 gene mutation provided by the invention and a 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 liquid phase chip for detecting BARD1 gene mutation has very good signal-to-noise ratio, no cross reaction exists between the designed probe and the anti-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, 5 mutant sites can be detected by one-step PCR to complete the amplification of 5 target sequences containing the mutant sites, and a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like are avoided, so that the detection accuracy can be greatly improved, and the accurate and simultaneously qualitative and quantitative analysis characteristics are embodied.
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 1 liquid phase chip for detecting mutation of BARD1 gene, mainly comprising:
first, ASPE primer
Specific primer sequences are designed aiming at wild types and mutant types of five common genotypes of T6883G, A43635G, G1670C, G25542A and G1134C of the BARD1 gene respectively. 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 BARD1 Gene
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 formulated with 10mmol/LTris Buffer as a 100pmol/mL stock solution.
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 10 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 10 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 arranging a spacer arm sequence with proper length between the anti-tag sequence and the microsphere, 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 (CH 2) n spacers (n.gtoreq.3), such as (CH 2) 12, (CH 2) 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 50ul of 0.1mol/L MES solution (pH 4.5) and 10ul of synthetic anti-tag molecule (100 nmol/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 (pH 8.0) ]]In 1mmol/LEDTA, and storing at 2-8 deg.C in dark.
Thirdly, amplifying the primer of the target sequence containing the mutation site
Aiming at five common genotypes of the BARD1 gene, T6883G, A43635G, G1670C, G25542A and G1134C, an amplification primer pair is designed (see Table 3), and 5 target sequences containing 5 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 prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Example 2 detection of samples Using the liquid phase chip for detecting mutation of BARD1 Gene described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH 5.0) formulation (250 ml):
2 XTM hybridization buffer
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
5 pairs of primers are designed, 5 target sequences respectively containing five common genotypes of the BARD1 gene, namely T6883G, A43635G, G1670C, G25542A and G1134C are amplified by multiple PCR in one step, the sizes of products are 369bp, 270bp, 276bp, 325bp and 313bp respectively, and the primer sequences (SEQ ID NO. 31-40) are shown in the table 3.
Firstly, preparing a multiplex PCR primer working solution: 100ul of primer stock solution of SEQ ID NO.31-40 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:
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 times SAP 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 was performed using the ASPE primer designed in example 1, and biotin-labeled dCTP was incorporated during the reaction, thereby allowing the product after the reaction to be 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:
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, 10 corresponding coated microspheres were selected per set (as described in example 1),the concentration of each microsphere is 2.5 multiplied by 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 2-5 min 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. The results of the measurements are shown in tables 4, 5 and 6.
The following requirements are placed on the fluorescence values (MFI) and data processing:
1. each locus needs to have at least one allele MFI greater than 300 and greater than 10 XPCR negative control MFI;
NET MFI = sample MFI-PCR negative control MFI (NET MFI less than 0 indicated as 0);
3. the mutation ratio is calculated according to the following formula from the data satisfying the above two conditions:
mutation ratio (mutant NETMFI + wild NETMFI)
4. A threshold (cut-off value) is empirically determined for the mutation ratio at each detection site to classify wild-type homozygotes, heterozygotes, and mutant homozygotes.
The method is used for detecting 20 samples of SNP sites of the BARD1 gene, and experimental data meet the requirements, so that the mutation ratio of the BARD1 gene can be calculated. The threshold value (cut-off value) is set as follows: the mutation ratio ranging from 0% to 20% is regarded as wild type homozygote; 30% -70% are considered heterozygotes; 80% -100% are considered variant homozygotes. The results of the sequencing method detection and the liquid phase chip are compared, and the coincidence rate of the detection results of the typing method provided by the invention is calculated. The coincidence rate of the BARD1 genotype detection result and the sequencing result of 20 samples detected by the method reaches 100%. The liquid phase chip for detecting the SNP of the BARD1 gene provided by the invention can accurately detect the SNP type of the BARD1, and the result is stable and reliable.
TABLE 4 sample test results (MFI)
TABLE 5 sample BARD1 Gene mutation ratio (%)
| Sample number | T6883G | A43635G | G1670C | G25542A | G1134C |
| 1 | 2% | 1% | 2% | 1% | 2% |
| 2 | 2% | 2% | 1% | 2% | 2% |
| 3 | 1% | 2% | 3% | 1% | 99% |
| 4 | 2% | 2% | 2% | 2% | 2% |
| 5 | 1% | 2% | 1% | 1% | 2% |
| 6 | 1% | 2% | 1% | 2% | 1% |
| 7 | 2% | 1% | 1% | 2% | 2% |
| 8 | 2% | 2% | 2% | 98% | 1% |
| 9 | 2% | 1% | 2% | 2% | 2% |
| 10 | 2% | 2% | 2% | 2% | 1% |
| 11 | 1% | 2% | 3% | 1% | 2% |
| 12 | 2% | 2% | 1% | 3% | 1% |
| 13 | 1% | 2% | 2% | 2% | 2% |
| 14 | 2% | 2% | 2% | 2% | 1% |
| 15 | 99% | 2% | 2% | 1% | 1% |
| 16 | 2% | 3% | 3% | 2% | 1% |
| 17 | 2% | 2% | 1% | 2% | 1% |
| 18 | 1% | 1% | 1% | 3% | 2% |
| 19 | 2% | 52% | 1% | 2% | 2% |
| 20 | 3% | 2% | 3% | 2% | 1% |
TABLE 6 analysis results of mutation types of sample BARD1 Gene
Example 3 detection of SNP site of BARD1 Gene by liquid chip of different ASPE primers
Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
Taking the liquid phase chip for detecting mutation at T6883G, A43635G, G25542A and G1134C sites of BARD1 genes as an example, specific primer sequences at the 3 'end of ASPE primers are designed aiming at wild types and mutant types of T6883G, A43635G, G25542A and G1134C respectively, Tag sequences at the 5' end of the ASPE primers are selected from SEQ ID NO.1-SEQ ID NO.10, and correspondingly, anti-Tag sequences which are coated on microspheres and are complementarily matched with the corresponding Tag sequences are selected from SEQ ID NO.21-SEQ ID NO. 30. 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
Firstly, 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 BARD1 Gene T6883G test results and Gene polymorphism analysis
Table 9 sample BARD1 Gene A43635G test results and Gene polymorphism analysis
Table 10 sample BARD1 Gene G25542A test results and Gene polymorphism analysis
Table 11 sample BARD1 Gene G1134C test results and Gene polymorphism analysis
As can be seen from the above examples, in other liquid phase chips for different mutation sites, the ASPE primers use different tag sequences, and the results are still stable and reliable, and specific data are omitted. When the tag sequence in the embodiment 1 is selected as the ASPE primer to match with the specific primer sequence, the effect is better (the signal to noise ratio is better), and the test group 1, the test group 5, the test group 8 and the test group 12 in the embodiment are referred to. 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.
Example 4 selection of primer sequences specific for the detection of mutation in BARD1 Gene
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking the polymorphic site detection liquid phase chip of BARD1 gene G1670C and G25542A as an example, taking the forward or reverse complementary sequence of the target sequence of the mutation site as a template, designing specific primer sequences at the 3' end of ASPE primers aiming at the wild type and the mutant type of G1670C and G25542A respectively, including the preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment 1 of the invention, as shown in Table 12. Wherein,
the internal base is a polymorphic site.
TABLE 12 specific primer sequences
Taking polymorphic site detection liquid phase chip of BARD1 gene G1670C and G25542A as an example, different specific primer sequences are selected for G1670C and G25542A, and tag sequence at 5' end of ASPE primer is fixed as best effect sequence in example 1, and anti-tag sequence corresponding to the best effect sequence is selected, and the specific design is shown in the following table (Table 13). 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 13 design of 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 14 sample BARD1 Gene G1670C test results and Gene polymorphism analysis
Table 15 sample BARD1 Gene G25542A test results and Gene polymorphism analysis
As can be seen from this example, when the specific primer sequence in example 1 is selected as the ASPE primer to match with the tag sequence, the effect is better (the signal to noise ratio is better), see test group 13 and test group 16 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 matching effect of the specific primer sequence and the different tag sequences in the embodiment 2 is still better, and the specific data are omitted.
Other specific primer sequences aiming at different SNP sites are matched with tag sequences, and the results are the same as those of the embodiment 2 and the embodiment, namely, the specific primer selected in the embodiment 1 has better signal to noise ratio, better detection effect and specific data are omitted.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A liquid phase chip for detecting BARD1 gene mutation is characterized by comprising:
(A) primer pairs of wild type and mutant ASPE designed for different mutation sites of BARD1 gene: 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.11 and SEQ ID NO.12 for the T6883G site, SEQ ID NO.13 and SEQ ID NO.14 for the A43635G site, SEQ ID NO.15 and SEQ ID NO.16 for the G1670C site, SEQ ID NO.17 and SEQ ID NO.18 for the G25542A site, and/or SEQ ID NO.19 and SEQ ID NO.20 for the G1134C site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 10;
(B) microspheres coated by different anti-tag sequences and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequences and the microspheres; the anti-tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30, 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.
2. The liquid phase chip for detecting BARD1 gene mutation of claim 1, wherein said amplification primers are: SEQ ID NO.31 and SEQ ID NO.32 at position T6883G, SEQ ID NO.33 and SEQ ID NO.34 at position A43635G, SEQ ID NO.35 and SEQ ID NO.36 at position G1670C, SEQ ID NO.37 and SEQ ID NO.38 at position G25542A, and/or SEQ ID NO.39 and SEQ ID NO.40 at position G1134C.
3. The liquid phase chip for detecting mutation of BARD1 gene according to claim 1 or 2, wherein said ASPE primer pair is the sequence consisting of SEQ ID No.1 and SEQ ID No.11 and the sequence consisting of SEQ ID No.2 and SEQ ID No.12 for the T6883G site, the sequence consisting of SEQ ID No.3 and SEQ ID No.13 and the sequence consisting of SEQ ID No.4 and SEQ ID No.14 for the A43635G site, the sequence consisting of SEQ ID No.5 and SEQ ID No.15 and the sequence consisting of SEQ ID No.6 and SEQ ID No.16 for the G1670C site, the sequence consisting of SEQ ID No.7 and SEQ ID No.17 and the sequence consisting of SEQ ID No.8 and SEQ ID No.18 for the G25542A site, and/or the sequence consisting of SEQ ID No.9 and SEQ ID No.19 and the sequence consisting of SEQ ID No.10 and SEQ ID No.20 for the G1134C site.
4. The liquid phase chip for detecting mutation in BARD1 gene according to claim 1 or 2, wherein the spacer arm has 5-10T.
5. Specific primers for BARD1 gene mutation detection are provided, wherein the specific primers are SEQ ID NO.11 and SEQ ID NO.12 for T6883G locus, SEQ ID NO.13 and SEQ ID NO.14 for A43635G locus, SEQ ID NO.15 and SEQ ID NO.16 for G1670C locus, SEQ ID NO.17 and SEQ ID NO.18 for G25542A locus, and/or SEQ ID NO.19 and SEQ ID NO.20 for G1134C locus.
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| WO2023025218A1 (en) * | 2021-08-25 | 2023-03-02 | 南京金斯瑞生物科技有限公司 | Primer set and gene chip method for detecting single base mutation |
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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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| WO2002059354A2 (en) * | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
| WO2005047533A1 (en) * | 2003-11-17 | 2005-05-26 | Tm Bioscience Corporation | Method of detecting mutations associated with thrombosis |
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