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Method for detecting (CAG)n repeated sequence by utilizing RNase H

A detection method and nucleotide sequence technology, applied in biochemical equipment and methods, biological testing, measuring devices, etc., can solve the problems of target DNA that cannot be reused, less fluorescent groups are difficult to detect, and detection accuracy is poor , to achieve the effect of enhanced detection sensitivity, high sensitivity and rapid detection

Active Publication Date: 2019-07-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many defects in these two methods. For example, because the target DNA cannot be reused, the fluorophores brought out are less and difficult to be detected, and cause problems such as poor detection accuracy and excessive waste.

Method used

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  • Method for detecting (CAG)n repeated sequence by utilizing RNase H
  • Method for detecting (CAG)n repeated sequence by utilizing RNase H
  • Method for detecting (CAG)n repeated sequence by utilizing RNase H

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Feasibility analysis of working principle:

[0046] (1) Dilute graphene oxide to 100 μg / ml with DEPC water, mix well, and set aside.

[0047] (2) Dissolve the fluorescently labeled aptamer R1 with DEPC water (sequence is 5'-FAM-GUCGUC GUC GUC GUCGUC GUC GUC GUC GUC-3') to prepare 20×10 -6 mol / L solution, mix well and set aside.

[0048] (3) Dissolve (CAG)n (sequence is 5'-CAGCAG CAG CAG CAG CAG CAG CAG CAG CAG CAG-3') with DEPC water to prepare 100×10 -6 mol / L solution, mix well and set aside.

[0049] (4) Dilute the fluorescently labeled aptamer R1 solution prepared in step (2) with DEPC water to 200×10 -9 mol / L, mix well, and become tube ①.

[0050] (5) Add the graphene oxide solution prepared in step (1) with a final concentration of 24 μg / ml into tube ①, mix well, and incubate at room temperature for 10 min to form tube ②.

[0051] (6) Add final concentration of 25×10 to tube ② -9 mol / L of (CAG)n prepared in step (3), mix well, incubate at room temperature for...

Embodiment 2

[0056] The ability of graphene oxide concentration to quench aptamer RNA fluorescence:

[0057] 1. Dilute graphene oxide to 100μg / ml with DEPC water and mix well.

[0058] 2. Dissolve the fluorescently labeled aptamer R1 (sequence is 5'-FAM-GUCGUC GUC GUC GUC GUCGUC GUC GUC GUC GUC-3') with DEPC water to prepare 20×10 -6 mol / L solution and mix well.

[0059] 3. Add different final concentrations of graphene oxide solution (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 28, 32 μg / ml) to Tris-HCl buffer solution respectively 20×10 in -6 mol / L FAM-R1, mix well, and incubate at room temperature for 30 min.

[0060] 4. Use a Hitachi F-7000 spectrofluorometer. The excitation wavelength was 480 nm, the excitation slit was 5 nm, the emission slit was 2.5 nm, the scanning speed was 1200 nm / min, and the fluorescence intensity at 520 nm was detected. The results showed that with the increase of graphene oxide concentration, the fluorescence intensity decreased continuously. After 24 μg / ...

Embodiment 3

[0063] To investigate the effect of aptamer fluorescence quenching time, the specific steps are as follows:

[0064] 1. Dilute graphene oxide to 100μg / ml with DEPC water and mix well.

[0065] 2. Dissolve the fluorescently labeled aptamer R1 (sequence is 5'-FAM-GUCGUC GUC GUC GUC GUCGUC GUC GUC GUC GUC-3') with DEPC water to prepare 20×10 -6 mol / L solution and mix well.

[0066] 3. Dissolve (CAG)n (sequence is 5'-CAGCAG CAG CAG CAG CAG CAG CAG CAG CAG CAG-3') with DEPC water to prepare 100×10 -6 mol / L solution and mix well.

[0067] 4. In containing 200×10 -9 Add 24 μg / ml graphene oxide solution to the Tris-HCl buffer solution of mol / L FAM-R1, mix well, and incubate at room temperature for 0, 2, 5, 8, 10, 12, 15, 20, 25, and 30 min respectively.

[0068] 5. Use a Hitachi F-7000 spectrofluorometer. The excitation wavelength was 480 nm, the excitation slit was 5 nm, the emission slit was 2.5 nm, the scanning speed was 1200 nm / min, and the fluorescence intensity at 520 nm wa...

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PUM

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Abstract

The invention discloses a method for detecting a (CAG)n repeated sequence by utilizing RNase H and belongs to the technical field of molecular biology. According to the method, a (CAG)n is mixed witha fluorescein-labeled complementary RNA aptamer; enzyme digestion reaction of restrictive endonuclease RNase H is executed: the mixed solution of (CAG)n and the aptamer with a solution of restrictiveendonuclease RNase H uniformly mixed for incubation; fluorescence detection is executed: graphene oxide is added into the solution subjected to the enzyme digestion reaction, uniformly mixing is executed for incubation to obtain a solution to be detected, and the fluorescence intensity is detected by using a fluorescence spectrophotometer. The method is simple to operate, rapid in detection and high in sensitivity, and the detection limit of (CAG)n can reach 1.12*10-10 mol / L, and it is detected that the DNA sequence containing (CAG)n has very high specificity.

Description

technical field [0001] The invention relates to a method for detecting (CAG) n repeating sequences by using RNase H, and belongs to the technical field of molecular biology. Background technique [0002] Neurological diseases such as Huntington's disease are neurodegenerative diseases that mainly affect motor function. The pathological changes are mainly the degeneration of the medium-sized spiny neurons in the striatum, especially the death of the striatal projection γ-aminobutyric acid neurons. The mutant gene is located on human chromosome 4, and there is a repeat sequence of trinucleotide CAG at the 17th codon downstream of the exon start codon ATG. Huntington's disease is caused by the abnormality of the CAG repeat sequence in this gene. Caused by extended mutations, and this repeated sequence is also considered a biomarker for the detection of the disease. [0003] Existing methods for detecting the repeating state of CAG sequences use nano-gold-DNA sensors or graphe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804G01N33/53
CPCC12Q1/6804G01N33/5308C12Q2563/107C12Q2521/301
Inventor 杨兆琪胡前行秦兰朱诗宇仇丽蓉苏咏欣金坚
Owner JIANGNAN UNIV
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