CN102226219A - Specific primer and liquid-phase chip for polymorphism detection in chromosome 6q23 section - Google Patents
Specific primer and liquid-phase chip for polymorphism detection in chromosome 6q23 section Download PDFInfo
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Abstract
The invention discloses a specific primer and a liquid-phase chip for polymorphism detection in a chromosome 6q23 section. The liquid-phase chip mainly comprises a specific primer, microspheres and amplified primers, wherein the specific primer comprises a tag sequence of a 5' terminal and a specific primer sequence of a 3' terminal for target gene polymorphism sites, wherein the specific primer is more than one pair of SEQ ID NO.15 and SEQ ID NO.16 for G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, and SEQ ID NO.27 and SEQ ID NO.28 for T135C; and the tag sequence is a sequence of SEQ ID NO.1 to SEQ ID NO.14. The prepared liquid-phase chip for polymorphism detection has a good signal-noise ratio, and the designed probe and an anti-tag sequence do not have cross reaction basically.
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 polymorphism of a chromosome 6q23 segment.
Background
Rheumatoid Arthritis (RA) is a systemic autoimmune disease with inflammatory invading joints and their surrounding connective tissue, and is also a polygenic genetic disease with a complex genetic pattern. Environmental factors and genetic factors influence the occurrence and development of RA.
At present, genome wide association analysis studies (GWAS) show that, as susceptibility genes related to RA, HLA (human leukocyte antigen) gene at 6p21.3, PTPN22 (non-receptor protein tyrosine phosphatase 22) gene at 1p23, 6q23 site, CTLA-4 (cytotoxic lymphocyte-associated antigen 4) gene, 9q33-34 site (including TRAF1 and C5 genes), STAT4 (signal transduction and transcription activator 4) gene at chromosome 2, and the like are predominant.
The invention mainly aims at 6q23 sites adjacent to OLIG3 (oligomer linkage transcription factor 3) and TNFAIP3(TNF-a-induced protein 3) genes, and TNFAIP3 has the function of negatively regulating and controlling inflammatory reaction caused by the down-regulation of the concentration of nuclear factor NF-kappa B. Mutations in the relevant sites of the 6q23 segment will affect the expression of the TNFAIP3 gene product and thus the risk of RA.
The mutation sites of the chromosome 6q23 segment detected by the target of the invention are shown as the following table:
| serial number | Content of site mutation in chromosome 6q23 segment | Shorthand writing |
| 1 | Nucleotide 107 of SEQ ID NO.57, the G → A mutation | G107A |
| 2 | At nucleotide 301 of SEQ ID NO.58, the G → A mutation occurred | G301A |
| 3 | Nucleotide 103 of SEQ ID NO.59, the G → A mutation | G103A |
| 4 | Nucleotide 219 of SEQ ID NO.60, the G → T mutation | G219T |
| 5 | Nucleotide 74 of SEQ ID NO.61, the T → C mutation | T74C |
| 6 | Nucleotide 163 of SEQ ID NO.62, the T → C mutation | T163C |
| 7 | Nucleotide 135 of SEQ ID NO.63, with the T → C mutation | T135C |
At present, there are a few methods for detecting and analyzing polymorphism of chromosome 6q23 segment gene, and there are generally: traditional solid phase chips and TaqMan technology, among others, the most commonly used method is TaqMan technology based on fluorescent quantitative PCR. The TaqMan technology has the advantages of simplicity and convenience in operation, rapidness in result, quantification and the like, but the TaqMan technology has the defects of easiness in sample pollution, easiness in cross reaction generation and high false positive rate, and meanwhile, the method has the limitation of detection flux, only one mutation type can be detected each time, and the requirement of practical application cannot be met.
Disclosure of Invention
One of the purposes of the present invention is to provide a liquid phase chip for detecting polymorphism of chromosome 6q23 segment, which can be used for detecting wild type and mutant type of 7 common genotypes G107A, G301A, G103A, G219T, T74C, T163C and T135C of chromosome 6q23 segment.
A liquid phase chip for detecting polymorphism of chromosome 6q23 segment mainly comprises:
A. wild type and mutant specific ASPE primers are respectively designed aiming at 7 common polymorphic sites of a chromosome 6q23 segment, each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence aiming at the polymorphic site of a target gene at the 3' end, and the specific primer sequences are respectively selected from: more than one pair of SEQ ID NO.15 and SEQ ID NO.16 for G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, SEQ ID NO.27 and SEQ ID NO.28 for T135C; the tag sequence is selected from the sequences in SEQ ID NO.1-SEQ ID NO. 14;
B. microspheres respectively coated with specific anti-tag sequences, wherein the anti-tag sequences can be correspondingly complementarily paired with the tag sequences selected in the A, the anti-tag sequences are selected from the sequences in SEQ ID NO.29-SEQ ID NO.42, a spacer arm sequence is further arranged between the anti-tag sequences and the microspheres, and each microsphere has different color codes;
C. primers for amplifying target sequences having one or more of polymorphic sites G107A, G301A, G103A, G219T, T74C, and T135C.
Preferably, the amplification primers are: more than one pair of SEQ ID NO.43 and SEQ ID NO.44 for G107A, SEQ ID NO.45 and SEQ ID NO.46 for G301A, SEQ ID NO.47 and SEQ ID NO.48 for G103A, SEQ ID NO.49 and SEQ ID NO.50 for G219T, SEQ ID NO.51 and SEQ ID NO.52 for T74C, SEQ ID NO.53 and SEQ ID NO.54 for T163C, SEQ ID NO.55 and SEQ ID NO.56 for T135C.
Preferably, the ASPE primers are: the sequence consisting of SEQ ID NO.1 and SEQ ID NO.15 and the sequence consisting of SEQ ID NO.2 and SEQ ID NO.16 for G107A, the sequence consisting of SEQ ID NO.3 and SEQ ID NO.17 and the sequence consisting of SEQ ID NO.4 and SEQ ID NO.18 for G301A, the sequence consisting of SEQ ID NO.5 and SEQ ID NO.19 and the sequence consisting of SEQ ID NO.6 and SEQ ID NO.20 for G103A, the sequence consisting of SEQ ID NO.7 and SEQ ID NO.21 and the sequence consisting of SEQ ID NO.8 and SEQ ID NO.22 for G219T, more than one pair of the sequence consisting of SEQ ID NO.9 and SEQ ID NO.23 and the sequence consisting of SEQ ID NO.10 and SEQ ID NO.24 for T74C, the sequence consisting of SEQ ID NO.11 and SEQ ID NO.25 and the sequence consisting of SEQ ID NO.12 and SEQ ID NO.26 for T163C, the sequence consisting of SEQ ID NO.13 and SEQ ID NO.27 and the sequence consisting of SEQ ID NO.14 and SEQ ID NO.28 for T135C.
Another objective of the invention is to provide a specific primer sequence for detecting polymorphism of chromosome 6q23 segment.
The technical scheme for realizing the purpose is as follows:
a specific primer sequence for detecting polymorphism of chromosome 6q23 segment, which is selected from the group consisting of: more than one pair of SEQ ID NO.15 and 16 for G107A, SEQ ID NO.17 and 18 for G301A, SEQ ID NO.19 and 20 for G103A, SEQ ID NO.21 and 22 for G219T, SEQ ID NO.23 and 24 for T74C, SEQ ID NO.25 and 26 for T163C, and SEQ ID NO.27 and 28 for T135C.
The main advantages of the invention are:
1. the coincidence rate of the detection result of the liquid phase chip provided by the invention and a sequencing method is up to 100%, and the time required by detection is far shorter than that of a common sequencing technology, thereby being particularly in line with the requirements of practical application. And (3) obtaining the liquid-phase chip system with the optimal combination through a large number of tests and reaction verification in a large number of specific primers. The prepared polymorphism detection liquid phase chip for the chromosome 6q23 segment has very good signal-to-noise ratio, cross reaction does not exist between the designed probe and the anti-tag sequence basically, the tag label sequence, the anti-tag label sequence are selected, and the tag label sequence is combined with a specific ASPE primer, so that the cross reaction can be avoided, and the parallel detection of a plurality of SNP 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 single site mutation condition, polymorphism conditions of a plurality of mutation sites can be detected simultaneously and parallelly, and detection effects are consistent.
3. The detection method of the invention has simple steps, and the 7 kinds of polymorphic sites can be detected by one-step multiplex PCR to complete the amplification of the target sequences containing 7 SNP sites, thereby avoiding a plurality of uncertain factors existing in the complex operation processes of repeated multiplex PCR and the like, greatly improving the detection accuracy and embodying the accurate and simultaneously 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 1 liquid phase chip for detecting polymorphism in chromosome 6q23 segment
Mainly comprises the following steps:
first, ASPE primer
Specific primer sequences were designed for wild-type and mutant of 7 common genotypes of chromosome 6q23 segment, G107A, G301A, G103A, G219T, T74C, T163C, and T135C, 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 chromosome 6q23 segment
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
According to the designed ASPE specific primer fragment, tag sequences are selected, secondary structures possibly formed among anti-tag sequences of the microspheres and between tag and the ASPE specific primer fragment are reduced to the maximum extent, and the number of the selected 14 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 14 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 (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. Preparing 10ng/ml of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) (from Pierce Chemical Co.) working solution), adding 2.5ul of EDC working solution into the microsphere suspension, incubating for 30 minutes at constant temperature, adding 2.5ul of EDC working solution, incubating for 30 minutes at constant temperature, washing once with 0.02% Tween-20 after the reaction is finished,and washed once more with 0.1% SDS solution. The washed microspheres coated with anti-tag sequence were resuspended in 100ul Tris-EDTA solution [10mmol/LTris (pH8.0) ]]In 1mmol/LEDTA, and storing at 2-8 deg.C in dark.
Thirdly, amplifying the primer of the target sequence containing the mutation site
Amplification primer pairs (see table 3) are designed aiming at 7 common genotypes G107A, G301A, G103A, G219T, T74C, T163C and T135C of the chromosome 6q23 segment, and 7 target sequences containing polymorphic sites are respectively amplified.
TABLE 3 primers for amplifying target sequences having polymorphic 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 chip for detecting chromosome 6q23 segment described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH5.0) formulation (250 ml):
| reagent | Origin of origin | Final concentration | The dosage of each 250ml |
| MES(2[N-Morpholino] | Sigma M-2933 | 0.05M | 2.44g |
| ethanesulfonic acid) | |||
| 5MNaOH | Fisher SS256-500 | --- | 5 drops of the Chinese traditional medicine |
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 at4 ℃.
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.
Secondly, PCR amplification of the sample to be detected
7 pairs of primers are designed, 7 common genotypes G107A, G301A, G103A, G219T, T74C, T163C and T135C respectively containing chromosome 6q23 segments are amplified by multiplex PCR in one step, the sizes of products are 464bp, 616bp, 351bp, 453bp, 278bp, 393bp and 345bp respectively, and the sequences of the primers (SEQ ID NO.43-56) are shown in the table 3.
Firstly, preparing a multiplex PCR primer working solution: 100ul of the primer stock solution of SEQ ID NO.43-56 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 at4 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 site to be detected into a 1.5ml microcentrifuge tube, 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 at4 deg.C for use.
Fifthly, hybridization reaction
1. Root of herbaceous plantAccording to the designed ASPE primers, 28 corresponding microspheres (each microsphere concentration is 2.5 multiplied by 10)5One/ml); each microsphere is respectively provided with different color codes, and the surface of each microsphere is respectively connected with a section of 24bp specific oligonucleotide sequence (anti-tag), and the anti-tag sequences can be respectively and specifically combined with tag sequences at the 5' ends of corresponding ASPE primers;
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. 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 NET MFI + wild-type NET MFI)
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.
By using the method to detect the polymorphic sites of the chromosome 6q23 segment in 20 samples, the experimental data meet the requirements, and therefore, the mutation ratio of the polymorphic sites 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 chromosome 6q23 segment type detection result and the sequencing result of 20 samples detected by the method reaches 100%. Therefore, the polymorphism detection liquid phase chip for the chromosome 6q23 segment can accurately detect the polymorphism site type of the chromosome 6q23 segment, 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 sample chromosome 6q23 segment mutation ratio (%)
TABLE 7 analysis of the mutation types of the sample chromosome 6q23 segment
| Sample number | Liquid phase chip detection result | Sequencing results |
| 1 | 163CC | 163CC |
| 2 | 219GT | 219GT |
| 3 | 74CC | 74CC |
| 4 | Wild type | Wild type |
| 5 | 107AA | 107AA |
| 6 | Wild type | Wild type |
| 7 | Wild type | Wild type |
| 8 | 301AA、103AA | 301AA、103AA |
| 9 | 135CC | 135CC |
| 10 | Wild type | Wild type |
| 11 | Wild type | Wild type |
| 12 | 163TC | 163TC |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | Wild type | Wild type |
| 16 | 107GA、219GT | 107GA、219GT |
| 17 | Wild type | Wild type |
| 18 | Wild type | Wild type |
| 19 | Wild type | Wild type |
| 20 | Wild type | Wild type |
Example 3 detection of polymorphic sites in chromosome 6q23 segment by liquid phase chip with 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 sites G301A and G219T of chromosome 6q23 segment as an example, specific primer sequences at the 3 'end of ASPE primers are designed aiming at wild type and mutant type of G301A and G219T respectively, the Tag sequence at the 5' end of the ASPE primers is selected from SEQ ID NO.1-SEQ ID NO.14, and correspondingly, the anti-Tag sequence coated on the microsphere and complementarily paired with the corresponding Tag sequence is selected from SEQ ID NO.29-SEQ ID NO. 42. The specific design is shown in the following table (table 8). 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 8 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 9 sample test results and Gene polymorphism analysis
TABLE 10 sample test results and Gene polymorphism analysis
In other liquid phase chips aiming at different mutation sites, the ASPE primers use different Tag sequences, the result is 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), see the test group 2 and the test group 5 in the embodiment. 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 polymorphism detection in chromosome 6q23 segment
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking the liquid phase chip for detecting mutation at sites G103A and T163C of chromosome 6q23 as an example, taking the forward or reverse complementary sequence of the target sequence of the mutation site as a template, specific primer sequences at the 3' end of ASPE primers are designed for the wild type and the mutant type of G103A and T163C respectively, including the preferred specific primer sequence and 2 alternative specific primer sequences in example 1 of the present invention, as shown in Table 11. Wherein,
the internal base is a polymorphic site.
TABLE 11 specific primer sequences
Taking the liquid phase chip for detecting mutations at G103A and T163C sites of chromosome 6q23 segment as an example, different specific primer sequences were selected for G103A and T163C, and the Tag sequence at the 5' end of the ASPE primer was fixed as the best effect sequence in example 1, and the anti-Tag sequence corresponding thereto was selected, and the specific design is shown in the following table (Table 12). 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 12 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 13 sample test results and analysis of Gene polymorphisms
TABLE 14 sample test results and analysis of Gene polymorphisms
As can be seen from this example, when the specific primer sequence in example 1 is selected as the primer for ASPE to match with the tag sequence, the effect is better (the signal to noise ratio is better), 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, and the matching effect of the specific primer sequence and the tag sequence in the embodiment 1 is still better. Other specific primer sequences aiming at different mutation 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 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 (6)
1. A liquid phase chip for detecting polymorphism of chromosome 6q23 segment is characterized by mainly comprising:
A. wild type and mutant specific ASPE primers designed respectively aiming at different polymorphic sites of a chromosome 6q23 segment, wherein each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence aiming at the polymorphic site of a target gene at the 3' end, and the specific primer sequences are respectively selected from the following groups: more than one pair of SEQ ID NO.15 and SEQ ID NO.16 for G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, SEQ ID NO.27 and SEQ ID NO.28 for T135C; the tag sequence is selected from the sequences in SEQ ID NO.1-SEQ ID NO. 14;
B. microspheres respectively coated with specific anti-tag sequences, wherein the anti-tag sequences can be correspondingly complementarily paired with the tag sequences selected in the A, the anti-tag sequences are selected from the sequences in SEQ ID NO.29-SEQ ID NO.42, a spacer arm sequence is further arranged between the anti-tag sequences and the microspheres, and each microsphere has different color codes;
C. primers for amplifying target sequences having one or more of polymorphic sites G107A, G301A, G103A, G219T, T74C, and T135C.
2. The liquid phase chip for detecting polymorphism of chromosome 6q23 segment according to claim 1, wherein the amplification primers are selected from the group consisting of: more than one pair of SEQ ID NO.43 and SEQ ID NO.44 for G107A, SEQ ID NO.45 and SEQ ID NO.46 for G301A, SEQ ID NO.47 and SEQ ID NO.48 for G103A, SEQ ID NO.49 and SEQ ID NO.50 for G219T, SEQ ID NO.51 and SEQ ID NO.52 for T74C, SEQ ID NO.53 and SEQ ID NO.54 for T163C, SEQ ID NO.55 and SEQ ID NO.56 for T135C.
3. The solution phase chip for detecting polymorphism of chromosome 6q23 segment according to claim 1, wherein the ASPE primers are selected from the group consisting of: the sequence consisting of SEQ ID No.1 and SEQ ID No.15 and the sequence consisting of SEQ ID No.2 and SEQ ID No.16 for G107A, the sequence consisting of SEQ ID No.3 and SEQ ID No.17 and the sequence consisting of SEQ ID No.4 and SEQ ID No.18 for G301A, the sequence consisting of SEQ ID No.5 and SEQ ID No.19 and the sequence consisting of SEQ ID No.6 and SEQ ID No.20 for G103A, the sequence consisting of SEQ ID No.7 and SEQ ID No.21 and the sequence consisting of SEQ ID No.8 and SEQ ID No.22 for G219T, more than one pair of the sequence consisting of SEQ ID NO.9 and SEQ ID NO.23 and the sequence consisting of SEQ ID NO.10 and SEQ ID NO.24 for T74C, the sequence consisting of SEQ ID NO.11 and SEQ ID NO.25 and the sequence consisting of SEQ ID NO.12 and SEQ ID NO.26 for T163C, the sequence consisting of SEQ ID NO.13 and SEQ ID NO.27 and the sequence consisting of SEQ ID NO.14 and SEQ ID NO.28 for T135C.
4. The liquid phase chip for detecting polymorphism of chromosome 6q23 according to claim 1, which mainly comprises:
A. specific ASPE primers: the sequence consisting of SEQ ID No.1 and SEQ ID No.15 and the sequence consisting of SEQ ID No.2 and SEQ ID No.16 for G107A, the sequence consisting of SEQ ID No.3 and SEQ ID No.17 and the sequence consisting of SEQ ID No.4 and SEQ ID No.18 for G301A, the sequence consisting of SEQ ID No.5 and SEQ ID No.19 and the sequence consisting of SEQ ID No.6 and SEQ ID No.20 for G103A, the sequence consisting of SEQ ID No.7 and SEQ ID No.21 and the sequence consisting of SEQ ID No.8 and SEQ ID No.22 for G219T, the sequence consisting of SEQ ID NO.9 and 23 and the sequence consisting of SEQ ID NO.10 and 24 for T74C, the sequence consisting of SEQ ID NO.11 and 25 and the sequence consisting of SEQ ID NO.12 and 26 for T163C, and the sequence consisting of SEQ ID NO.13 and 27 and the sequence consisting of SEQ ID NO.14 and 28 for T135C;
B. microspheres coated with specific anti-tag sequences respectively, wherein the anti-tag sequences can be correspondingly complementary and matched with the tag sequences selected in the A, the anti-tag sequences are selected from the sequences in SEQ ID NO.29-SEQ ID NO.42, and spacer arm sequences are further arranged between the anti-tag sequences and the microspheres;
C. the amplification primers are as follows: SEQ ID NO.43 and SEQ ID NO.44 for G107A, SEQ ID NO.45 and SEQ ID NO.46 for G301A, SEQ ID NO.47 and SEQ ID NO.48 for G103A, SEQ ID NO.49 and SEQ ID NO.50 for G219T, SEQ ID NO.51 and SEQ ID NO.52 for T74C, SEQ ID NO.53 and SEQ ID NO.54 for T163C, and SEQ ID NO.55 and SEQ ID NO.56 for T135C.
5. The liquid phase chip for detecting polymorphisms of chromosome 6q23 according to any one of claims 1 to 4, wherein the spacer sequence is 5 to 10T.
6. A specific primer for detecting polymorphism of chromosome 6q23 segment, which is characterized in that the sequence of the specific primer is as follows: more than one pair of SEQ ID NO.15 and SEQ ID NO.16 for G107A, SEQ ID NO.17 and SEQ ID NO.18 for G301A, SEQ ID NO.19 and SEQ ID NO.20 for G103A, SEQ ID NO.21 and SEQ ID NO.22 for G219T, SEQ ID NO.23 and SEQ ID NO.24 for T74C, SEQ ID NO.25 and SEQ ID NO.26 for T163C, and SEQ ID NO.27 and SEQ ID NO.28 for T135C.
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| CN101988897A (en) * | 2009-08-07 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Liquid phase chip detector based on quantum dot |
| CN102031286A (en) * | 2010-06-08 | 2011-04-27 | 广州益善生物技术有限公司 | Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer |
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| CN101633963A (en) * | 2009-08-11 | 2010-01-27 | 广州益善生物技术有限公司 | Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof |
| CN102031286A (en) * | 2010-06-08 | 2011-04-27 | 广州益善生物技术有限公司 | Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer |
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