Specific detection primers and detection liquid phase chip for chromosome Xp11.22 segment polymorphism
A polymorphism detection and liquid phase chip technology, applied in the field of molecular biology, can solve the problems of detection limitations, high false positive rate, complicated operation, etc., and achieve consistent detection results, good detection specificity, and low cross-reaction rate Effect
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Embodiment 1
[0041] Example 1 The liquid chip for detecting mutations in the chromosome Xp11.22 segment mainly includes:
[0042] 1. ASPE Primers
[0043] Specific primer sequences were designed for the wild-type and mutant types of three common genotypes A65G, G121A and C162T in the chromosome Xp11.22 segment. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0044] Table 1 ASPE primer sequence (tag sequence + specific primer sequence) of chromosome XP11.22 segment
[0045]
[0046] Each ASPE primer consists of two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.
[00...
Embodiment 2
[0058] Example 2 Using the chromosome Xp11.22 segment mutation detection liquid chip described in Example 1 to detect samples, the formulations of the various solutions are as follows:
[0059] 50mM MES buffer (pH5.0) formula (250ml):
[0060]
[0061] 2×Tm hybridization buffer
[0062]
[0063]
[0064] Store at 4°C after filtration.
[0065] ExoSAP-IT kit was purchased from US USB Company.
[0066] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0067] 1. Sample DNA extraction:
[0068] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0069] 2. PCR amplification of samples to be tested
[0070] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes A65G, G121A and C162T respectively in the chromosome Xp11.22 segment. NO.19-24) see Table 3 above.
[0071] First prepare the multiplex P...
Embodiment 3
[0112] Example 3 Detection of the SNP site of chromosome Xp11.22 segment by liquid chip with different ASPE primers
[0113] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
[0114] Taking the liquid-phase chip for detection of site mutations in the A65G, G121A and C162T segments of chromosome Xp11.22 as an example, the specific primer sequences of the 3' ends of the ASPE primers were designed for the wild-type and mutant types of A65G, G121A and C162T, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.6. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 an...
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