Probes, primers and reagent kits for detecting five mutations of human gene TP53
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A technology for detecting probes and kits, which is applied in the biological field and can solve problems such as easy contamination of samples, difficulty in differentiation, and poor accuracy
Active Publication Date: 2016-04-06
武汉海吉力生物科技有限公司
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[0005] In order to solve the problems of the existing TP53 gene mutation detection methods, such as low detection efficiency, easy sample contamination, long detection time, poor accuracy, and difficulty in discrimination, the present invention provides primers and probes for detecting five mutations of the human TP53 gene
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Embodiment 1
[0078] 1. Primers and probes of the detection kit for detecting 7 mutations of human TP53 gene:
[0079] According to the wild-type sequence of the TP53 gene (NCBIReferenceSequence: NM_000546.5) published in the NCBI database, the R175H (TM1) mutation site in exon 5, the R248W (TM2) and R248Q (TM3) mutation sites in exon 7, and the exon 8 Using exon R273C (TM4) and R273H (TM5) mutation sites as reference, design specific R175H mutation primers and probes (see Table 2), specific R248W mutation primers and probes (see Table 3), specific R248Q mutation Primers and probes (see Table 4), specific R273C mutation primers and probes (see Table 5) and specific R273H mutation primers and probes (see Table 6). Using the mutant plasmid and wild-type plasmid constructed by genetic engineering as templates, a real-time fluorescent PCR mutation detection system was established to achieve high sensitivity and high specificity detection of TP53 gene mutation.
[0080] (1) Specific primer and ...
Embodiment 2
[0156] The human TP53 gene 5 mutation detection kit of the present invention is used to detect clinically collected tissue samples.
[0157] In this example, tissue samples from patients diagnosed as breast cancer were collected and genomic DNA was extracted from them. The TP53 gene mutation detection kit was used to detect the mutation status of the TP53 gene in the samples to be tested. Specific steps are as follows:
[0158] (1) Sample genomic DNA extraction
[0159] Paraffin-embedded tissue samples were extracted using QIAampDNAFFPETissueKit (CatNo.56404). Extract the genomic DNA of the sample to be tested according to the instructions, and use the ultraviolet spectrophotometer to detect the concentration and DNA quality of the extracted DNA sample. The concentration of DNA is required to be ≥10ng / μL, and the quality of DNA OD260 / 280 is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2-10 ng / μL with TE (10 mmol / L, pH 8.0) as a PCR template fo...
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Abstract
The invention discloses probes, primers and reagent kits for detecting five mutations of a human gene TP53. According to five primers and probes for TM1-TM5, each mutation primer can be combined with a conserved sequence of the gene TP53, the primer for the mutation ARMS can be specifically combined with the corresponding mutation sequence, and the mutation sequence is amplified. The detection probe can be combined with amplified fragments, the blocking probe can be specifically combined with a wild type sequence corresponding to a mutation site, and wild type non-specificity amplification is restrained. The specificity mutation primers and the probe blocking technology are adopted, and the five mutation types of the gene TP53 can be accurately detected; mutations of the gene TP53 are rapidly detected conveniently through the built reagent kits of a real-time fluorescence PCR amplified reaction system, operation is easy, and the result is easy to read. The detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, 20 ng wild type genomic DNA non-specificity amplification is achieved, and the detectability reaches up to 1%.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting five mutations of human TP53 gene. Background technique [0002] The wild-type TP53 gene is a tumor suppressor gene, and the p53 protein encoded by it can repair damaged cells or remove severely damaged cells so as to avoid the harm of these cells to the body. TP53 gene mutations are related to 50% of human tumors, currently found are liver cancer, breast cancer, bladder cancer, gastric cancer, colon cancer, prostate cancer, soft tissue sarcoma, ovarian cancer, brain tumor, lymphocytic tumor, esophageal cancer, lung cancer, Osteogenic sarcoma, etc., and there are differences in TP53 allele mutations in different tumors. [0003] TP53 mutations in human tumors are mainly in highly conserved regions, most of which are missense mutations, among which the mutations at codon sites such as 175, 245, 248, 249, 273, and 283 are the highest, which may be...
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