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Preparation method of multi-fluorescent nucleic acid probe and application thereof

A fluorescent nucleic acid and probe technology, which is applied in the field of preparation of multi-fluorescent nucleic acid probes, can solve the problems of limiting the sensitivity of probes and affecting the detection limit of biosensors, and achieves enhanced sensitivity, short time required, and simple preparation methods Effect

Pending Publication Date: 2021-07-23
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are several classic fluorescent nucleic acid probes including molecular beacon nucleic acid probes, strand displacement nucleic acid probes, nucleic acid aptamer probes, etc., and their signal output depends on a single fluorescent group at the end of the probe. , that is, the ratio of probe to fluorophore is 1:1, which limits the sensitivity of the probe to a certain extent and affects the detection limit of related biosensors.

Method used

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  • Preparation method of multi-fluorescent nucleic acid probe and application thereof
  • Preparation method of multi-fluorescent nucleic acid probe and application thereof
  • Preparation method of multi-fluorescent nucleic acid probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 prepares the multi-fluorescence nucleic acid probe method for detecting EV-71 virus

[0022] A. Preparation of double-stranded DNA:

[0023] Template sequences, upstream primers, and downstream primers were all synthesized by Takara Biological Company.

[0024] Template sequence: 5'-GAG CAG TCA CAG TCC AGA AGG GCA TGT CAG GGC TTG GAT ACCTCG CAT TCA CCC TTG CAC GAT AC-3';

[0025] Upstream primer: 5'-GAG CAG TCA CAG TCC AGA AG-3' (phosphorylated at the 5' end)

[0026] Downstream primer: 5’-GTA TCG TGC AAG GGT GAA TGC-3’

[0027] PCR reaction system

[0028]

[0029]

[0030] The PCR reaction was carried out according to the above PCR reaction system, the amplification conditions were: 95°C, 30s; 55°C, 30s; 72°C, 30s, after 40 cycles, 72°C, 5min. The amplified product is purified by the kit to obtain pure double-stranded DNA.

[0031] B. Preparation of probe single strand:

[0032] The double-stranded DNA obtained in step A was mixed with Lambda ...

Embodiment 2

[0037] Comparison of the double-stranded DNA synthesized by embodiment 2EdUTP and dTTP

[0038] According to step A in Example 1, replace EdUTP with dTTP to obtain double-stranded DNA. The double-stranded DNA, the high-density alkyne double-stranded DNA synthesized by EdUTP obtained in Example 1, the purified high-density alkyne double-stranded DNA, and the probe chain were analyzed by agarose gel electrophoresis. Proceed as follows:

[0039] Gel production: Add accurately weighed agarose powder to a conical flask with 60mL of 1X TBE buffer, heat and dissolve in a microwave oven, cool to about 50-60°C, add ethidium bromide, and mix well Introduce into the glue mold and let the glue solidify at room temperature.

[0040] Sample loading: Take an appropriate amount of sample and mix with 6× sample buffer, and the sample volume for each sample tank is 10 μL.

[0041] Electrophoresis: After adding the sample, close the cover of the electrophoresis tank and turn on the power imme...

Embodiment 3

[0043] The comparison of embodiment 3 synthetic probe strands and standard probes

[0044] The purchased standard probe sequence is: 5'-GTA TCG TGC AAG GGT GAA TGC GAG GTA TCC AAGCCC TGA CAT GCC CTT CTG GAC TGT GAC TGC TC-3', a fluorescent molecule is attached to the 5' end;

[0045] The sequence of the multi-fluorescent nucleic acid probe synthesized by the present invention is: 5'-GTA TCG TGC AAG GGT GAA TGC GAGGTA TCC AAG CCC TGA CAT GCC CTT CTG GAC TGT GAC TGC TC-3', and there are multiple fluorescent molecules attached to the DNA chain;

[0046] When the two probes were diluted to the same concentration of single-stranded DNA, both at 32ng / μL, the absorbance of cy3 at 550nm was measured by Nanodrop 2000, and the results were as follows figure 2 shown.

[0047] figure 2 The schematic diagram of the ultraviolet absorption spectrum shown is compared with the absorbance value of cy3 at 550nm, and the fluorescent molecule of the multi-fluorescent nucleic acid probe synthes...

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Abstract

The invention relates to the technical field of nucleic acid detection material preparation, in particular to a preparation method and an application of a multi-fluorescent nucleic acid probe, 5-ethynyl-uracil deoxynucleotide (5-EdUTP) is used for replacing thymine deoxynucleotide (dTTP) for nucleic acid amplification, and high-density alkyne double-stranded DNA is obtained; after the obtained double-stranded DNA is treated by an excision enzyme Lambda Exonuclease, the 5'end phosphorylation labeled nucleic acid chain is digested, and the high-density alkyne single-stranded DNA is obtained; through a click reaction catalyzed by copper ions, a dye cy3-azide with an azide group is successfully modified to a single-chain DNA of high-density alkyne, and the multi-fluorescent nucleic acid probe is obtained. The fluorescent groups of the multi-fluorescent nucleic acid probe prepared by the invention are increased, signal amplification is realized structurally, the sensitivity of the fluorescent nucleic acid probe in researches such as analysis and detection, biological imaging and the like can be enhanced, and the practical value of the fluorescent nucleic acid probe is improved.

Description

technical field [0001] The invention relates to the technical field of preparation of nucleic acid detection materials, in particular to a preparation method and application of a multi-fluorescent nucleic acid probe. Background technique [0002] Nucleic acid probe refers to a nucleic acid fragment with a known sequence and a marker, which can hybridize with its complementary nucleic acid sequence and is used for the detection of a specific gene sequence in a sample to be tested. Or, in the presence of molecules that specifically interact with it, the configuration and conformation of the nucleic acid probe will change, thereby causing a signal change, which can be used for targeted molecular recognition in the fields of chemistry, biology, medicine, and pharmacy. Among the numerous nucleic acid probes, fluorescent nucleic acid probes have become one of the research hotspots in analytical chemistry because of their high sensitivity, good specificity, diverse designs, and str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C12Q1/686C12Q1/6806
CPCC09K11/06C12Q1/686C12Q1/6806C09K2211/1466C12Q2563/107
Inventor 郝先余蒙蒙杨一飞邱壮刘小榕贺超楠刘婷
Owner NANCHANG UNIV
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