Detection method of microRNA (micro Ribonucleic Acid)

A detection method and polyuracil technology, applied in the field of microRNA detection, can solve the problem that the detection technology has not yet been reported, and achieve the effect of low background

Inactive Publication Date: 2016-06-08
吴滨
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, the combination of in vitro polymerized nucleotide amplification reaction and the syn...

Method used

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  • Detection method of microRNA (micro Ribonucleic Acid)
  • Detection method of microRNA (micro Ribonucleic Acid)
  • Detection method of microRNA (micro Ribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Double polymerase extension of uracil sequence: mix microRNA and template DNA (pDNA), anneal and hybridize at 75°C for 15 minutes, then slowly cool to room temperature, add polymerase-containing Klenow fragment (KFexo - ), deoxyuridine triphosphate and terminal deoxynucleotidyl transferase (buffer solution, reacted in a water bath at 35°C for 4 hours to prepare a polyuracil base sequence.

[0020] (2) Synthesis of fluorescent copper nanoclusters: Add ascorbate to the polyuracil base sequence solution in step (1), then add CuCl 2 , and react at room temperature for 10 minutes to prepare red fluorescent copper nanoclusters with polyuracil base sequence as template.

[0021] Depend on figure 1 A shows that when microRNA-21 exists, the CuNCs synthesized in situ using the polyuracil base sequence formed by the extension of uracil by double polymerases as a template has a strong fluorescence emission peak at 600nm (curve a), and the optimal excitation wavelength is 340n...

Embodiment 2

[0027] According to the research process of Example 1, the microRNA detection method was determined.

[0028] (1) Double polymerase extension of uracil sequence: Mix microRNA with known content and template DNA (pDNA), anneal and hybridize at 75°C for 15 minutes, then slowly cool to room temperature, add polymerase-containing Klenow fragment (KFexo - ), a buffer solution of deoxyuridine triphosphate and terminal deoxynucleotidyl transferase (TdTase), and react in a water bath at 35°C for 4 hours to prepare a polyuracil base sequence;

[0029] (2) Synthesis of fluorescent copper nanoclusters: add ascorbate to the polyuracil base sequence solution in step (1), and then add CuCl 2 , reacting at room temperature for 10 minutes, making red fluorescent copper nanoclusters with polyuracil base sequence as template, and obtaining a solution containing red fluorescent copper nanoclusters;

[0030] (3) Use a fluorescence spectrophotometer to calibrate the standard relationship curve be...

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Abstract

The invention discloses a detection method of microRNA (micro Ribonucleic Acid), and belongs to the field of optical biosensing techniques. A to-be-detected sample is mixed with template DNA (Deoxyribonucleic Acid), an obtained first mixture is annealed and hybridized, a buffer solution containing polymerase Klenow fragment, deoxyuracil nucleoside triphosphate and terminal deoxynucleotidyl transferase is added into a reaction product, an obtained second mixture is caused to react for 4h at 35 DEG C in a water bath, so as to prepare a polyuracil base sequence; sodium ascorbate is added into a polyuracil base sequence solution obtained in the step (4), and then CuCl2 is added into an obtained third mixture, and an obtained fourth mixture is caused to react for 10min at a room temperature, so as to prepare a red-fluorescence copper nano cluster which uses the polyuracil base sequence as a template, thereby obtaining a solution containing the red-fluorescence copper nano cluster. The intensity of the red fluorescence of the solution including the red-fluorescence copper nano cluster is measured by adopting a spectrofluorophotometer; by comparing the result about the intensity of the red fluorescence with a standard relation curve, the concentration of the microRNA is calculated and obtained.

Description

technical field [0001] The invention relates to a method for detecting microRNA, which belongs to the technical field of optical biosensing. Background technique [0002] Mature microRNAs are a class of single-stranded, non-coding protein, short endogenous RNAs (approximately 19-23 nucleotides in length). As an important post-transcriptional regulator of gene expression, MicroRNA can regulate the splitting or translational repression of messenger ribonucleic acid. MicroRNA plays an important role in many biological processes, including not only early diagnosis and prognostic indicators of cancer, but also interventional therapy and discovery of cancer drugs. Therefore, it is necessary to develop microRNA detection methods with high sensitivity and good selectivity. [0003] In vitro polymerized nucleotides are widely used in biological analysis as a powerful tool due to their remarkable signal amplification ability. In the study of microRNA detection methods, in vitro pol...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2521/101C12Q2521/131C12Q2563/137C12Q2525/207
Inventor 吴滨
Owner 吴滨
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