Preparation method of human specific gene fluorescent probe and probe prepared by method

A fluorescent probe, specific technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome preparation process of fluorescent probes, low labeling efficiency, and unstable labeling. , to achieve the effect of stable labeling method, simplified preparation process and high efficiency

Active Publication Date: 2021-03-16
河南赛诺特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for preparing a human-specific gene fluorescent probe and the probe prepared by the method, so as to solve the problems of cumbersome preparation process, low labeling efficiency and unstable labeling in the prior art.

Method used

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  • Preparation method of human specific gene fluorescent probe and probe prepared by method
  • Preparation method of human specific gene fluorescent probe and probe prepared by method
  • Preparation method of human specific gene fluorescent probe and probe prepared by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of human CDKN2A gene fluorescent probe

[0042] 1) Find the specific gene sequence

[0043] Find the CDKN2A gene on the UCSC website (http: / / genome.ucsc.edu / index.html), extend the length of the upstream and downstream of the gene location (chr9:21,967,753-994,624) by 100kb, and shield the repetitive sequence. Part of the non-repeated sequence of the post-CDKN2A gene such as figure 1 As shown, where "N" means a masked repeat sequence, and non-N means a non-repeat sequence (the complete sequence is shown in other supporting documents);

[0044] 2) Design specific primers

[0045] Referring to the design of DNA primers, search for the specific primer sequence on the obtained non-repetitive sequence, which requires that the specific primer sequence length is 20-30bp, the Tm value is 60-62°C, the distance between the two specific primers is at least 5bp, and the specific primer sequence The quantity is 1744 pieces. Import the designed specific pri...

Embodiment 2

[0065] Embodiment 2: Detection of human CDKN2A gene by fluorescence in situ hybridization (FISH)

[0066] 1) Sample processing:

[0067] The peripheral blood cell droplet samples were soaked in 2×SSC solution at room temperature for 2 minutes, then soaked in 70%, 90%, and 100% ethanol at room temperature for 2 minutes to dehydrate, took out the slides, and dried them at room temperature for later use.

[0068] 2) Denatured hybridization of the sample with the probe:

[0069] Take 10ul of the probe working solution prepared in Example 1 and add it dropwise to the hybridization area, cover the cover glass while avoiding air bubbles, seal the slide along the edge of the cover glass with rubber glue, and place the slide on a hybridization instrument at 90°C Denaturation for 2 minutes, hybridization at 55°C for 30 minutes-3 hours.

[0070] 3) Washing after hybridization:

[0071] Remove the slide, remove the rubber and remove the coverslip. Place the slides in 73°C preheated wa...

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Abstract

The invention discloses a preparation method of a human specific gene fluorescent probe and the probe prepared by the method. According to the preparation method of the fluorescent probe provided by the invention, by extending the upstream and downstream of the position of a specific gene by 1000kb and shielding repeated sequences, primers are designed for non-repeated sequences of the specific gene, ensuring the specific primer sequence length of 20 to 30 bp, a Tm value of 60-62 DEG C, a separation between the two specific primers at least 5 bp, and the number of specific primers at least 1000, then a universal primer sequence 5'-TGTAAAACGACGGCCAGT-3' is added to the 5' end of the primers, and the primers are synthesized. The universal primer at the 5' end of the primers can be hybridizedwith a universal probe with a fluorophore so as to realize detection of human specific genes. The method is simple in steps and high in labeling stability and efficiency, and the preparation processof the specific gene fluorescent probe is greatly simplified. The human CDKN2A gene fluorescent probe prepared by the method can be used for accurately and quickly detecting the human CDKN2A gene.

Description

technical field [0001] The invention relates to a method for preparing a human-specific gene fluorescent probe and the probe prepared by the method, belonging to the field of molecular pathological diagnosis. Background technique [0002] Fluorescence in situ hybridization (FISH) is a molecular genetics technique developed on the basis of radioactive in situ hybridization. The principle is to use fluorescently labeled DNA probes to perform in situ hybridization with the DNA of the sample to be tested, and to distinguish and count the fluorescent signals under a fluorescent microscope to detect the presence and abundance of a specific DNA. At present, for the preparation of human-specific gene fluorescent probes, the labeling method of BAC clone nick translation is mostly used. BAC cloning nick translation method requires complex preparatory work such as cell activation, shaking bacteria, and BAC clone extraction in the early stage. Therefore, the labeling method using the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6811C12Q1/6841C12N15/11
CPCC12Q1/6886C12Q1/6811C12Q1/6841C12Q2600/156C12Q2531/113C12Q2525/151C12Q2563/107Y02A50/30
Inventor 霍清园李贵喜李三华齐华王少辉李艳敏胡娟张绍敏刘文弟
Owner 河南赛诺特生物技术有限公司
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