A method for detecting (CAG) n repeat sequences using rnase H

A nucleotide sequence and kit technology, applied in the field of molecular biology, can solve the problems of non-reusable target DNA, difficult detection due to few fluorophores, poor detection accuracy, etc., and achieves enhanced detection sensitivity, high sensitivity, Detect fast effects

Active Publication Date: 2021-01-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for detecting (CAG) n repeat sequences using rnase H
  • A method for detecting (CAG) n repeat sequences using rnase H
  • A method for detecting (CAG) n repeat sequences using rnase H

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Working principle Feasibility analysis:

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

[0047] (2) Dissolve fluorescently labeled aptamer R1 (sequence 5'-FAM-GUCGUC GUC GUC GUCGUC GUC GUC GUC GUC GUC-3') with DEPC water, and 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-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 a tube ①.

[0050] (5) Add the graphene oxide solution prepared in step (1) at 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 a final concentration of 25×10 to tube ② -9 mol / L (CAG)n prepared in step (3), mix well, incubate at room temperature for 30 min, ...

Embodiment 2

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

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

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

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

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

Embodiment 3

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

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

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

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

[0067] 4. In the containing 200×10 -9 Add 24 μg / ml graphene oxide solution to the mol / L Tris-HCl buffer solution of 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 fluorescence spectrophotometer. The excitation wavelength is 480nm, the excitation slit is 5nm, the emission slit is 2.5nm, the scanning speed is 1200nm / min, and the fluorescence intensity at 520nm is ...

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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 repeat sequence by using RNase H, belonging 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 striatal medium-sized spiny neurons, especially the death of striatal projection γ-aminobutyric acid neurons. The mutant gene is located on human chromosome 4, and there is a trinucleotide CAG repeat sequence 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 expansion mutations, and this repeated sequence is also considered as a biomarker for the detection of the disease. [0003] Existing methods for detecting the repetition of CAG sequences use nano-gold-DNA sensors or graphene oxide-DNA sensors. Thes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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