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Fluorescent chemical sensor and detection method for detecting oxidative damage in telomeres and application of fluorescent chemical sensor and detection method

A chemical sensor and internal oxidation technology, which is applied in the field of biological detection and molecular biology, can solve problems such as difficult to quantify 8-oxoG damage, and achieve the effect of improving detection sensitivity, improving sensitivity, and high sensitivity

Active Publication Date: 2019-08-20
SHANDONG NORMAL UNIV
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Problems solved by technology

However, the inventors discovered that they require specific damage markers and sophisticated thermal cyclers to precisely control thermal cycling
Adap, a derivative of adenosine triphosphate, can be used to distinguish 8-oxoG damage from normal G bases in human telomere sequences, but this method is difficult to quantify 8-oxoG damage

Method used

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  • Fluorescent chemical sensor and detection method for detecting oxidative damage in telomeres and application of fluorescent chemical sensor and detection method
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  • Fluorescent chemical sensor and detection method for detecting oxidative damage in telomeres and application of fluorescent chemical sensor and detection method

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Embodiment 1

[0072] 1. Detection principle and method steps of the present invention:

[0073] Genomic DNA was first extracted by digestion with dsDNA fragmentase to obtain DNA with 50-200 bp. In the human genome, telomeres are composed of repetitive sequences (TTAGGG) n Composition, which forms long regions of double-stranded DNA terminating in single-stranded 3'G-rich overhangs. To enrich telomeres, 3'-biotinylated capture probes were designed to hybridize to telomeres and streptavidin-coated magnetic beads were used to separate telomeres from genomic DNA. All isolated telomeres were end-labeled with ddCTP by polymerization using TdT to prevent non-specific amplification. Subsequently, in order to improve the efficiency of the base excision repair reaction, an auxiliary probe modified with ddC at the 3' end was also designed to hybridize to single-stranded telomeres to form formamide pyrimidine [fapy]-DNA glycosylase (Fpg) dsDNA substrate.

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Abstract

The invention provides a fluorescent chemical sensor and detection method for detecting oxidative damage in telomeres and an application of the fluorescent chemical sensor and the detection method. Specificity of a repair enzyme and polymerization mediated by terminal deoxynucleotidyl transferase are combined so as to start a cycle cutting signal probe induced by endonuclease IV, induce an AP sitein an Endo IV splitting signal probe, cause release of AF488 fluorophore on the signal probe, and besides, generate a signal probe having free 3'-OH end. Each signal probe having free 3'-OH can be effectively extended through TdT to generate a long poly-A sequence. The released poly-A sequence and a newly-generated poly-A sequence can induce the repeated cycle cutting of the signal probe so as torelease more AF488 fluorophore. Under magnetic separation, the fluorophore in supernatant can be imaged and quantified by unimolecule. The fluorescent chemical sensor and detection method have favorable actual application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection and molecular biology, and in particular relates to a fluorescent chemical sensor for detecting oxidative damage in telomere, a detection method and application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Telomeres consist of a double-stranded repeat sequence (TTAGGG)n with single-stranded overhanging ends. Telomeres seal the ends of eukaryotic chromosomes and play key roles in genome stability and cell viability. The G-rich nature of the TTAGG G repeat makes telomeres highly susceptible to oxidative damage, especially in the single-stranded region, since it is more r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825
CPCC12Q1/6825C12Q2521/531C12Q2521/301C12Q2521/131C12Q2563/107C12Q2523/303
Inventor 张春阳张艳华若男相东雪
Owner SHANDONG NORMAL UNIV
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