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Kit for detecting mutations of A-G at 1555th site and C-T at 1494th site of mitochondrial gene
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A technology of mitochondrial genes and kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., to achieve the effects of simple sample processing, strong specificity, and accurate quantification
Active Publication Date: 2015-02-18
FUJIAN ACAD OF MEDICAL SCI
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[0005] At present, there is no product on the market that uses pyrosequencing technology for mitochondrial gene mutation detection
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Embodiment 1
[0028] 1. Design and synthesis of primers and probes
[0029] Studies have shown that the mutation sites closely related to drug-induced deafness are 1555 and 1494. Primers were designed using PyroMark Assay Design2.0 software; the amplification primers and sequencing primers were first purified by PAGE and then purified by HPLC, and the 5' end of SEQ ID NO.2 was biotin-labeled.
[0030] The sequences of amplification primers and sequencing primers are:
[0031] Forward amplification primer SEQ ID NO.1: 5'-GGCCCTGAAGCGCGTACACA-3' (mtDNA nt1463-1482);
[0032] Reverse amplification primer SEQ ID NO.2: 5'-biotin-TGGGTTTGGGGCTAGGTTTA-3'(mtDNA nt1673-1692), the amplified product is 230bp;
[0033] Sequencing primer 1 SEQ ID NO.3: 5'-GCCCTGAAGCGCGTACACAC-3' (mtDNA nt1464-1483), used to detect the mitochondrial gene C1494T mutation;
[0034] Sequencing primer 2 SEQ ID NO.4: 5'-TACGCATTTATATAGAGGAG-3' (mtDNA nt1535-1554), used to detect the mitochondrial gene A1555G mutation.
[...
Embodiment 2
[0036] 1. Sample testing
[0037] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, UNG enzyme, and Taq DNA polymerase, aliquot the system, add sample DNA, and a blank control as templates to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure. The main components of the 50 μL reaction system are as follows: 45 μL of PCR reaction solution, 0.05 μL of UNG enzyme, 0.5 μL of Taq enzyme, 1 μL of forward and reverse amplification primers, 2 μL of template, and the rest is ultrapure water.
[0038] The PCR reaction program of this system: (1) 95°C, 5min, 1 cycle; (2) 95°C, 30s, 50°C, 30s, 72°C, 30s, 30 cycles; (3) 72°C, 7min, 1 cycle Cycle; (4) 4°C, holding, 1 cycle.
[0039] After the amplification is completed, the PCR results are detected by agarosegel electrophoresis f...
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Abstract
The present invention provides a kit for detecting the mutation of A-G at position 1555 and C-T at position 1494 of mitochondrial gene. The kit includes the PCR reaction containing the forward amplification primer shown in SEQ ID NO.1 and the reverse amplification primer shown in SEQ ID NO.2 Liquid, sequencing primer 1 and sequencing primer 2 shown in SEQIDNO.3 and SEQIDNO.4. PCR combined with pyrosequencing technology is used to detect mitochondrial gene mutations in the patient's blood DNA. The kit of the present invention can monitor the reaction process in real time, the reaction time is short, and the PCR product can be sequenced on a pyrosequencer after simple treatment. The operation is simple, the reaction time is short, and the throughput is high. in mutation analysis.
Description
technical field [0001] The invention relates to the field of in vitronucleic acid detection, which uses PCR combined with pyrosequencing technology to detect mitochondrial gene mutations in blood DNA of patients, and specifically relates to a kit for detecting mutations of 1555 A-G and 1494 C-T of mitochondrial genes. Background technique [0002] Mitochondrial 12S rRNA gene is a mutational hotspot in aminoglycoside antibiotic-induced non-syndromic hearing loss. Since the A1555G and C1494T mutations form G-C and U-A pairings in the 12S rRNA gene, it is easier to bind to aminoglycosideantibiotics, which is why people with these mutations will develop or aggravate deafness when they are exposed to aminoglycosideantibiotics. Therefore, the detection of these two point mutations has important clinical significance for the prevention of deafness caused by aminoglycoside antibiotic toxicity. At present, there are many methods for the detection of mitochondrial deafness genes, ...
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