Excision repair cross complementation group 2 (ERCC2) gene mutation detection specific primers and liquid chip kit
A kit and detection solution technology, applied in the field of molecular biology, can solve problems such as the use of antibodies, the lack of uniform standards for judging the results of operation steps, affecting promotion and application, and detection limitations, so as to avoid uncertain factors and improve detection accuracy , The detection effect is consistent
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Embodiment 1
[0023] The ERCC2 gene mutation detection liquid chip described in this embodiment mainly includes:
[0024] 1. ASPE Primers
[0025] Specific primer sequences were designed for wild-type and mutant types of four common genotypes of ERCC2 gene, G934A, T128G, C2133T and A396C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:
[0026] ASPE primer sequence (tag sequence+specific primer sequence) of table 1 ERCC2 gene
[0027]
[0028]Each ASPE primer includes 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 the above table 1). Among them, the bases marked in the "box" were used as the target to detect the wild-type and mutant bases of the mutation site, and all ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized ...
Embodiment 2
[0040] Example 2 Detection of samples using the ERCC2 gene mutation detection liquid chip described in Example 1
[0041] The formula of described various solutions is as follows:
[0042] 50mM MES buffer (pH5.0) formula (250ml):
[0043]
[0044] 2×Tm hybridization buffer
[0045]
[0046] Store at 4°C after filtration.
[0047] ExoSAP-IT kit was purchased from US USB Company.
[0048] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0049] 1. Sample DNA extraction:
[0050] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.
[0051] 2. PCR amplification of samples to be tested
[0052] Four pairs of primers were designed, and multiplex PCR amplified four target sequences containing four target detection mutation sites G934A, T128G, C2133T and A396C of the ERCC2 gene in one step. The product sizes were 334bp, 284bp, 301bp and 381bp, respectively. SEQ ID NO.25...
Embodiment 3
[0094] The liquid phase chip kit of embodiment 3 different ASPE primers detects the SNP site of ERCC2 gene
[0095] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
[0096] Taking the liquid-phase chip for detecting mutations at the G934A, T128G and C2133T sites of the ERCC2 gene as an example, the specific primer sequences at the 3' end of the ASPE primers were designed for the wild-type and mutant types of G934A, T128G and C2133T, respectively, and the Tag sequence at the 5' end of the ASPE primers It is selected from SEQ ID NO.1-SEQ ID NO.8, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.7-SEQ ID NO.24. 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 and Example 2....
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