SDA reaction solution for microRNA SDA detection based on agncs/hpDNA probe

A reaction liquid, -CCCTTAATCCCC-3 technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as difficulty in ensuring specificity, shorten reaction time, and save reaction materials. Effect

Active Publication Date: 2018-12-18
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although simple hybridization detection is simple, it is difficult to ensure good specificity.

Method used

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  • SDA reaction solution for microRNA SDA detection based on agncs/hpDNA probe
  • SDA reaction solution for microRNA SDA detection based on agncs/hpDNA probe
  • SDA reaction solution for microRNA SDA detection based on agncs/hpDNA probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, detection scheme feasibility verification

[0051] (1) AgNCs / HpDNAs probe synthesis

[0052] The synthesis was carried out according to the method in Yeh. HpDNAs (RED 16(7s)C), AgNO 3 , NaBH 4 The starting concentrations were 100 μM, 1 mM and 1 mM, respectively. The stock concentration of phosphate buffer was 200mM (Pi, pH8.0). Equimolar AgNO 3 and NaBH 4 According to 1RED 16(7s)C:25AgNO 3 :25NaBH 4 The proportion of the three was added to HpDNA, so that the final concentrations of the three were 15μM, 375μM and 375μM (Pi, 20mM, pH8.0). Among them, NaBH 4 It needs to be freshly prepared, and finally quickly added to the Ag+ / HpDNA mixture within 30s, and then shake vigorously for 45s~1min. The resulting solution was placed at room temperature in a dark environment for 18 hours to obtain stable AgNCs / HpDNAs probes.

[0053] (2) Verification of fluorescence enhancement effect of G-rich sequence hybridization

[0054] To the obtained probe AgNCs / ...

Embodiment 2

[0057] Embodiment 2, single miRNA detection

[0058] (1) AgNCs / RED 16(7s)C probe synthesis

[0059] Prepare the AgNCs / RED 16(7s)C probe as described in (1), but the final concentration of RED 16(7s)C is 5 μM, AgNO 3 and NaBH 4 The amount added is still according to 1RED 16(7s)C:25AgNO 3 :25NaBH 4 conduct.

[0060] (2) Single miRNA detection

[0061]Each reaction tube contains 50 μL SDA reaction solution, which contains the following components: 1×Nb2.1 self-made buffer (buffer pH7.9@25°C) (50mM NaAc, 10mM Tris-HAc, 10mM Mg(Ac) 2 and 100μg / mL BSA) 200μM dNTPs, 10UBsu polymerase (without DTT), AgNCs / RED 16(7s)C (2.5μM HpDNA), different concentrations of target miRNA (0.05-2.5μM miR-16-5p) and 2.5μM Primer Pri 2. The obtained reaction solution was incubated at 55°C for 55min, and then stored in a dark environment at 4°C, then the fluorescence detection could be performed on a fluorescence spectrophotometer, and the experiment was repeated 3 times.

[0062] (3) Results ...

Embodiment 3

[0065] Embodiment 3, single base mismatch nucleic acid detection

[0066] (1) Single base mismatch nucleic acid detection

[0067] According to the steps described in (1, 2) in Example 2, only the nucleic acid to be tested was changed to 0.5 μM mismatched nucleic acid, that is, the AgNCs / RED 16(7s)C probe was used to detect miR-19b-3p, h16DMⅠ (sequence SEQ ID NO: 5) and h16DMII (SEQ ID NO: 6).

[0068] (2) Results

[0069] Mismatched nucleic acid detection results such as Figure 4 shown. It can be seen that in the detection based on the AgNCs / RED 16(7s)C probe, only miR-16-5p (λex=580nm) showed the highest fluorescence enhancement signal, and the fluorescence enhancement of other samples at this excitation wavelength was uniform. Weaker, especially, h16DMⅠ showed fluorescence quenching under excitation of 580nm wavelength. In summary, the detection based on AgNCs / RED 16(7s)C probe showed good specificity.

[0070] The results of gel electrophoresis showed that the mis...

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Abstract

The invention discloses a microRNA SDA detecting method based on AgNCs / HpDNA probes. The method is characterized in that hairpin type DNA-templated silver nanoclusters are used as novel molecular beacons; in a chain-substitute isothermal amplification reaction mediated by primer dangling end rich-G sequences, single detection of gastric cancer plasma microRNA markers is achieved by the aid of rich-G fluorescence enhancement effects generated by hybridization. The method is high in specificity, short in reaction time, low in material use amount and simple in operation procedures, and a new direction for creating a fast and simple novel microRNA detecting method is developed.

Description

technical field [0001] The invention relates to the fields of medicine and molecular diagnosis, and is a microRNA SDA detection method based on AgNCs / HpDNA probes, in particular to a single method for synthesizing silver nanocluster probes binding strands to replace isothermal amplification based on hairpin DNA templates. microRNA detection method. Background technique [0002] MicroRNA (miRNA) is a class of endogenous non-coding small RNA molecules with a length of about 21 nt, which can participate in the regulation of mRNA expression levels through precise or non-precise complementary pairing with mRNA. Multiple miRNAs can coordinately regulate one mRNA, or one miRNA can also affect multiple target genes at the same time, thus forming a highly complex regulatory network that affects a series of biological functions from molecule to cell to tissue level. Abnormal expression of miRNA is closely related to disease and cancer. Most importantly, it has been found that a vari...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2525/207C12Q2525/301C12Q2531/119C12Q2563/107
Inventor 崔大祥张晶璞
Owner SHANGHAI JIAOTONG UNIV
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