Kit and method for detecting mutation of TERT promoter through pyrosequencing method

A pyrosequencing method and kit technology, applied in the field of molecular biology, can solve problems such as poor prognosis, affecting activity, and increasing malignancy of the disease, and achieve high-throughput detection, simple operation, and small sample volume.

Inactive Publication Date: 2018-07-17
XIANGYA HOSPITAL CENT SOUTH UNIV
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Problems solved by technology

The mutation located in the TERT promoter region can affect the activity of TERT, and then affect the function of telomerase, thereby promoting the

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  • Kit and method for detecting mutation of TERT promoter through pyrosequencing method

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Embodiment Construction

[0026] The present invention will be described in detail below in conjunction with examples.

[0027] Detection of TERT by Pyrosequencing C228T Kit, comprising: PCR buffer solution, high-fidelity TaqDNA polymerase, primers, template DNA, pyrosequencing kit, magnetic beads, binding buffer solution, washing buffer solution, annealing buffer solution and denaturation buffer solution; the primers consist of Composition of PCR amplification primers and sequencing primers: TERT C228T / C250T Upstream primer 5′-CACAGACGCCCAGGACCG-3′,

[0028] TERT C228T / C250T The downstream primer 5′-GCGCTGCCTGAAACTCGC-3′, 5′ was biotinylated,

[0029] TERT C228T / C250T Sequencing primer 5'-CCCGCCCCGTCCCGA-3'.

[0030] The above kit and detection method will be described in detail below in conjunction with the examples.

[0031] 1. DNA extraction

[0032] 1.1 The preparation and inspection of reagent materials before the experiment are as follows:

[0033] (1) Check the shelf life of the kit and...

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Abstract

The invention discloses a kit and a method for detecting the mutation of a TERT promoter through a pyrosequencing method. The mutation of the TERT promoter specifically refers to mutations of -124bp 1295, 228C>T and -146bp 1259, 250C>T on the upstream of the promoter. The kit comprises a PCR (Polymerase Chain Reaction) buffer solution, a TaqDNA polymerase, a primer, a template DNA, a pyrosequencing kit, a magnetic bead, a combined buffer solution, a washing buffer solution, an annealing buffer solution and a denaturation buffer solution. The kit can accurately efficiently detect the mutation of the TERT promoter at high throughput, so that the evidence is provided for the early diagnosis and screening of the thyroid cancer.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a kit and a method for detecting TERT promoter mutation by pyrosequencing. Background technique [0002] Telomeres are small nucleoprotein structures at the ends of linear chromosomes that maintain genome stability by preventing DNA fusion and degradation. In normal cells, telomeres gradually shorten with cell mitosis, thereby regulating cell aging and apoptosis. However, in cancer cells, telomerase is activated and continuously expressed, maintaining the length of telomeres and making tumor cells immortal. Telomerase is a ribonucleoprotein that uses its own RNA as a template to synthesize telomere DNA, add it to the end of chromosomes, and extend telomeres. Telomerase is highly expressed in a variety of tumor cells and plays an important role in the proliferation and differentiation of cancer cells. Inhibiting the activity of telomerase c...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301
Inventor 李智唐新月石峰
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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