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Gene detection kit including nucleic acid molecules of universal fluorescent subtracted probe and application thereof

A detection kit and gene detection technology, applied in the field of gene detection, can solve the problems of complicated operation, difficult to popularize in a large area, and difficult to standardize hybridization conditions.

Active Publication Date: 2018-12-07
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional FISH technology is complicated to operate, has many influencing factors, takes a long time, is technically difficult, and is difficult to promote on a large scale.
Other subtractive probe technologies require the synthesis of specific fluorescently labeled probes and fluorescently quenched probes for each gene, which is costly and difficult to standardize hybridization conditions.

Method used

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  • Gene detection kit including nucleic acid molecules of universal fluorescent subtracted probe and application thereof
  • Gene detection kit including nucleic acid molecules of universal fluorescent subtracted probe and application thereof
  • Gene detection kit including nucleic acid molecules of universal fluorescent subtracted probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 Gene detection method of the present invention

[0074] 1. The composition of the detection kit

[0075] Schematic as figure 1 Shown:

[0076] A sequence:

[0077] 5′–CTGAAGCGCTTCGCGAGCCM 1 m 2 N 1 N 2 N 3 N 4 N 5 …N 6 N 7 N 8 N 9 N 10 m 3 m 4 CTGAAGCGCTTCGCGAGCC-3'; wherein, N 1 N 2 N 3 N 4 N 5 …N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene to be tested or its fragment, M 1 m 2 and M 3 m 4 is the linker sequence;

[0078] B sequence: 5′-n 5 no 4 no 3 no 2 no 1 m 2 m 1 TAGGCTCGCGAAGC-3'; n1 no 2 no 3 no 4 no 5 with N 1 N 2 N 3 N 4 N 5 The sequence complement of m 1 m 2 with M 1 m 2 sequence complementary;

[0079] C sequence: 5′-GCGAAGCGCTTCAGm 4 m 3 no 10 no 9 no 8 no 7 no 6 -3'; n 6 no 7 no 8 no 9 no 10 with N 6 N 7 N 8 N 9 N 10 The sequence complement of m 3 m 4 with M 3 m 4 sequence complementary;

[0080] D sequence: 5′.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3′

[0081] E ...

Embodiment 2

[0089] Embodiment 2 Gene detection method of the present invention

[0090] 1. The composition of the detection kit

[0091] Schematic as figure 2 Shown:

[0092] ⑴A sequence:

[0093] 5'–CTGAAGCGCTTCGCGAGCCM 1 m 2 N 1 N 2 N 3 N 4 N 5 …N 6 N 7 N 8 N 9 N 10 m 3 m 4 CTGAAGCGCTTCGCGAGCC-3'; wherein, N 1 N 2 N 3 N 4 N 5 …N 6 N 7 N 8 N 9 N 10 is the complementary sequence of the gene to be tested or its fragment, M 1 m 2 and M 3 m 4 is the linker sequence;

[0094] Among them, M 1 m 2 for TA or AC, and / or, M 3 m 4 It is AT, CA or TC.

[0095] ⑵B sequence: 5'-n 5 no 4 no 3 no 2 no 1 m 2 m 1 TAGGCTCGCGAAGC-3'; n 1 no 2 no 3 no 4 no 5 with N 1 N 2 N 3 N 4 N 5 The sequence complement of m 1 m 2 with M 1 m 2 sequence complementary;

[0096] CDC sequence: 5'-GCGAAGCGCTTCAGm 4 m 3 no 10 no 9 no 8 no 7 no 6 -3'; n 6 no 7 no 8 no 9 no 10 with N 6 N 7 N 8 N 9 N 10 The sequence complement of m 3 m 4 with M 3 m 4 se...

experiment example 1

[0123] Experimental example 1 adopts the method of the present invention to detect brain cell edema gene AQP4 gene

[0124] 1. Detection method

[0125] The sample to be tested is the brain tissue nerve cells of rats with cerebral edema.

[0126] The inventive method detects according to the method of embodiment 1:

[0127] (1) First, design and synthesize a set of fluorescence subtraction probes according to the mRNA sequence of the AQP4 gene, and its sequences and labels are respectively:

[0128] A sequence:

[0129] 5′-.CTGAAGCGCTTCGCGAGCCTAGCTACATGGAGGTGGAGGACAACCGATCTGAAGCGCTTCGCGAGCC-3′

[0130] Sequence B: 5′.-CCATGTAGCTAGGCTCGCGAAGC-3′

[0131] C sequence: 5'.-GCGAAGCGCTTCAGATCGGTTGTC-3'

[0132] D sequence: 5′.-FAM-GGCTCGCGAAGCGCTTCAG–FAM-3′

[0133] E sequence: 5'-.TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3';

[0134] (2) Dissolve the above-mentioned probes in TE buffer respectively, so that the concentration of sequence A is 1umol / L, the concentration of sequence B is...

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Abstract

The invention discloses a gene detection kit and a gene detection method. According to the fluorescent subtracted probe technology, fluorescence of a fluorescent labeled probe not hybridized with a target nucleic acid is quenched by a quenching gene of a universal quenching probe, so that non-hybrid fluorescent signals can be removed by controlling the hybridizing temperature without tedious washing steps which are difficult to control, so that the effects that the nucleic acid hybridizing efficiency is improved greatly, the time and labor are saved, fluorescence in situ hybridization can be finished quickly, and the specific genes or expression distribution thereof are detected are achieved. The kit is an improvement of conventional FISH, and is excellent in application prospect.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a gene detection kit containing a universal fluorescence subtraction probe nucleic acid molecule and an application thereof. Background technique [0002] Nucleic acid molecular hybridization means that two nucleic acid single strands with certain homology can be combined into double strands according to base complementarity under certain conditions (suitable temperature and humidity, ionic strength, etc.). Nucleic acid molecular hybridization can be divided into Southern hybridization, Northern hybridization, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), chip hybridization (belonging to solid-liquid phase hybridization) and so on. Each hybridization technique has its own characteristics, and many related techniques can be derived due to the different selection of probes, which play an important role in different fields. [0003] FISH is a non-radioactive ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2563/107
Inventor 李孝锦夏庆杰康焰
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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