CN113150769A - Preparation method and application of multi-fluorescent nucleic acid probe - Google Patents
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1441—Heterocyclic
- C09K2211/1466—Heterocyclic containing nitrogen as the only heteroatom
Abstract
The invention relates to the technical field of nucleic acid detection material preparation, in particular to a preparation method and application of a multi-fluorescence nucleic acid probe, wherein 5-ethynyl-uracil deoxynucleotide (5-EdUTP) is used for replacing thymine deoxynucleotide (dTTP) to carry out nucleic acid amplification to obtain double-stranded DNA of high-density alkyne; treating the obtained double-stranded DNA with Exonuclease Lambda Exonuclease, and digesting the nucleic acid chain which is phosphorylated and marked at the 5' end to obtain high-density alkyne single-stranded DNA; through a copper ion catalyzed click reaction, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne to obtain the multi-fluorescent nucleic acid probe. The fluorescence groups of the multi-fluorescence nucleic acid probe prepared by the invention are increased, the signal amplification is realized structurally, the sensitivity of the fluorescence nucleic acid probe in the research of analysis detection, biological imaging and the like can be enhanced, and the practical value of the fluorescence nucleic acid probe is improved.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection material preparation, in particular to a preparation method and application of a multi-fluorescence nucleic acid probe.
Background
The nucleic acid probe refers to a nucleic acid fragment with known sequence and a marker, and can be hybridized with a nucleic acid sequence complementary to the nucleic acid fragment for detecting a specific gene sequence in a sample to be detected. Or the configuration and conformation of the nucleic acid probe can be changed in the presence of molecules with specific interaction with the nucleic acid probe, so that signal change is caused, and the nucleic acid probe can be used for target molecule recognition in the fields of chemistry, biology, medicine, pharmacy and the like. Among many nucleic acid probes, fluorescent nucleic acid probes have the characteristics of high sensitivity, good specificity, design diversification, strong quantitative analysis capability and the like, and thus become one of the research hotspots of analytical chemistry. The fluorescent nucleic acid probe mainly comprises a target recognition unit and a signal transduction unit. The functionalized nucleic acid is one of ideal recognition units of the fluorescent nucleic acid probe, consists of a nucleic acid segment with a known sequence, and has the advantages of good stability, strong binding force, good biocompatibility, easy synthesis and modification and the like. The fluorescent reporter unit is usually an organic dye molecule or an inorganic fluorescent nanomaterial and is responsible for converting the change in chemical environment caused by the binding of the recognition element to the analyte into a detectable signal during the detection process.
Several classical fluorescent nucleic acid probes exist, such as molecular beacon type nucleic acid probes, strand displacement type nucleic acid probes, and aptamer probes, and the signal output of the two probes depends on a single fluorophore at the end of the probe, namely, the ratio of the probe to the fluorophore is 1: 1, which limits the sensitivity of the probe to some extent, affecting the detection limit of the relevant biosensor.
Disclosure of Invention
The invention aims to provide a method for synthesizing a multi-fluorescent nucleic acid probe, which is simple, easy to manufacture and suitable for common laboratories.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of a multi-fluorescent nucleic acid probe comprises the following steps:
A. carrying out nucleic acid amplification by using 5-ethynyl-uracil deoxynucleotide (5-EdUTP) to replace thymine deoxynucleotide (dTTP) to obtain double-stranded DNA of high-density alkyne;
B. treating the double-stranded DNA obtained in the step A by Exonuclease Lambda Exonuclease, and digesting the nucleic acid chain of the 5' end phosphorylation marker to obtain high-density alkyne single-stranded DNA, namely a probe chain;
C. through a copper ion catalyzed click reaction, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne to obtain the multi-fluorescent nucleic acid probe.
Further, the PCR reaction system for nucleic acid amplification in the step A is as follows: 1 XPCR Buffer (Mg2+ Plus), 1. mu.M forward and 1. mu.M downstream primers, 20 pg/100. mu.L Template, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM EdUTP, 2.5 units/100. mu. LTaKaRa Taq enzyme, the remainder being ddH2O; carrying out PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: at 95 ℃ for 30 s; 30s at 55 ℃; at 72 ℃ for 30s, and after 40 cycles, at 72 ℃ for 5 min; and purifying the amplification product by using a kit to obtain pure double-stranded DNA.
Further, in the step B, the double-stranded DNA and the Lambda Exonuclease Exonuclease are mixed according to the proportion of 2 mu g/10U, and the incubation conditions are as follows: 30min at 37 ℃; 75 ℃ for 10 min.
Further, the conditions for the click reaction in step C were 60. mu.L of a single-stranded DNA solution, 20. mu.L of 100mM cy3-azide, 10. mu.L of 0.1M PBS buffer, pH7.4, 10. mu.L of CuAAC catalyst solution, and incubation at 37 ℃ for 60 min. The preparation method of the CuAAC catalytic liquid comprises the following steps: 70 mu L H2O，4μL 100mM THPTA，1μL 100mM CuSO425 μ L of 100mM sodium ascorbate solution was mixed well and left at room temperature for 20 min.
The invention also provides an application of the multi-fluorescence nucleic acid probe, which is used in a biosensor, such as a graphene-nucleic acid probe biosensor, wherein the multi-fluorescence nucleic acid probe is adsorbed on the surface of a graphene nanosheet, and fluorescence is quenched due to fluorescence resonance energy transfer between a fluorescence molecule and the nanosheet; when the detected target nucleic acid is combined with the probe to form a double chain, namely, the double chain is desorbed from the surface of the graphene, the fluorescence resonance energy transfer disappears, the fluorescence is recovered, and a process that the fluorescence is from nothing to nothing is realized. The target is quantitatively analyzed according to the proportion of the recovery intensity of fluorescence to the concentration of the target, and the sensitivity of the fluorescent probe is improved by orders of magnitude compared with that of the traditional fluorescent probe.
Compared with the prior art, the invention has the beneficial effects that:
(1) the prepared multi-fluorescent nucleic acid probe has a plurality of fluorescent groups on one probe chain, so that signal amplification is realized structurally, and the sensitivity is enhanced;
(2) the preparation method of the multi-fluorescent nucleic acid probe is simple, few in synthesis steps and short in required time, and can be quickly synthesized in a common laboratory.
(3) The multi-fluorescence nucleic acid probe prepared by the invention can be widely applied to various fields such as biosensors and biological imaging.
Drawings
FIG. 1 is an agarose gel electrophoresis image during probe synthesis;
FIG. 2 is a schematic diagram of the UV absorption spectra of the synthesized probe strand and a standard probe.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 method for preparing a Polyfluorescent nucleic acid Probe for detecting EV-71 Virus
A. Preparation of double-stranded DNA:
the template sequence, the upstream primer and the downstream primer are all synthesized by Takara Bio Inc.
Template sequence: 5'-GAG CAG TCA CAG TCC AGA AGG GCA TGT CAG GGC TTG GAT ACC TCG CAT TCA CCC TTG CAC GAT AC-3', respectively;
an upstream primer: 5'-GAG CAG TCA CAG TCC AGA AG-3' (5 ' end phosphorylation)
A downstream primer: 5'-GTA TCG TGC AAG GGT GAA TGC-3'
PCR reaction system
Carrying out PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: at 95 ℃ for 30 s; 30s at 55 ℃; 72 ℃ for 30s, 40 cycles, 72 ℃ for 5 min. And purifying the amplification product by using a kit to obtain pure double-stranded DNA.
B. Preparation of a single probe strand:
mixing the double-stranded DNA obtained in the step A with Lambda Exonuclease Exonuclease according to the proportion of 2 mu g/10U, wherein the incubation conditions are as follows: 30min at 37 ℃; 75 ℃ for 10 min. Digesting the nucleic acid chain of the 5' end phosphorylation marker, and purifying to obtain high-density alkyne single-chain DNA;
the sequence of the probe strand is as follows: 5'-GTA TCG TGC AAG GGT GAA TGC GAG GTA TCC AAG CCC TGA CAT GCC CTT CTG GAC TGT GAC TGC TC-3'
C. Ligation of single-stranded DNA with multiple fluorescent molecules:
preparing a CuAAC catalytic liquid: 70 μ L H20, 4 μ L THPTA (100mM), 1 μ L CuSO4(100mM), 25. mu.L of sodium ascorbate solution (100mM) was mixed well and left at room temperature for 20 min; 60 muL of the single-stranded DNA solution, 20. mu.L of cy3-azide (100mM), 10. mu.L of PBS (0.1M, pH7.4), 10. mu.L of the catalytic solution of LCuAAC, was incubated at 37 ℃ for 60 min. And purifying by using a kit to obtain the pure multi-fluorescent nucleic acid probe.
D. And adsorbing the multi-fluorescence nucleic acid probe on the surface of the graphene nanosheet to prepare the biosensor of the graphene-nucleic acid probe. Fluorescence resonance energy transfer occurs between the fluorescent molecules and the nanosheets, so that fluorescence is quenched; when the detected target nucleic acid is combined with the probe to form a double chain, namely, the double chain is desorbed from the surface of the graphene, the fluorescence resonance energy transfer disappears, the fluorescence is recovered, and a process that the fluorescence is from nothing to nothing is realized. The target is quantitatively analyzed according to the proportion of the recovery intensity of fluorescence to the concentration of the target, and the sensitivity of the fluorescent probe is improved by orders of magnitude compared with that of the traditional fluorescent probe.
Example 2 comparison of EdUTP with dTTP synthesized double-stranded DNA
Double-stranded DNA was obtained by replacing EdUTP with dTTP according to the procedure A in example 1. The double-stranded DNA, the high-density alkyne double-stranded DNA synthesized with EdUTP obtained in example 1, the purified high-density alkyne double-stranded DNA, and the probe strand were analyzed by agarose gel electrophoresis. The method comprises the following steps:
preparing glue: adding agar sugar powder accurately weighed into a conical flask added with 60mL of 1 XTBE buffer solution, heating and dissolving in a microwave oven, cooling to about 50-60 ℃, adding ethidium bromide, fully mixing uniformly, introducing into a gel-making mold, and solidifying the gel at room temperature.
Loading: an appropriate amount of sample was mixed with 6 Xloading buffer, and the loading amount per sample well was 10. mu.L.
Electrophoresis: after the sample is added, the electrophoresis tank cover is closed, and the power supply is immediately switched on. The voltage was controlled to be kept at 90V, and when the band was moved to about 2cm from the front edge of the gel, the electrophoresis was stopped, and a photograph was taken under UV light and observed, resulting in FIG. 1.
FIG. 1 is an agarose gel electrophoresis of the individual DNA strands of step A, B, 1 being a double strand synthesized at A, G, C, T four nucleotides; 2 is high-density alkyne double-stranded DNA synthesized by EdUTP; 3 is purified high-density alkyne double-stranded DNA; 4 is high-density alkyne single-stranded DNA obtained after digestion by exonuclease, namely a probe chain. The result of the agarose gel electrophoresis picture shows that EdUTP can successfully replace dTTP to synthesize double-stranded DNA, and the subsequent exonuclease digestion process is carried out to successfully synthesize a single probe chain.
Example 3 comparison of synthetic Probe strands with Standard probes
The standard probe sequences purchased were: 5'-GTA TCG TGC AAG GGT GAA TGC GAG GTA TCC AAG CCC TGA CAT GCC CTT CTG GAC TGT GAC TGC TC-3', a fluorescent molecule is connected to the 5 ' end;
the sequence of the multi-fluorescent nucleic acid probe synthesized by the invention is as follows: 5'-GTA TCG TGC AAG GGT GAA TGC GAG GTA TCC AAG CCC TGA CAT GCC CTT CTG GAC TGT GAC TGC TC-3', DNA multiple fluorescent molecules are linked to the chain;
when both the probes were diluted so that the concentrations of the single-stranded DNAs were identical and both were 32 ng/. mu.L, the absorbance at 550nm of cy3 was measured by Nanodrop 2000, and the results are shown in FIG. 2.
FIG. 2 is a schematic diagram of an ultraviolet absorption spectrum showing that the synthesized multi-fluorescent nucleic acid probe of the present invention has 4.6 times as much fluorescent molecules as the purchased standard probe, as compared with the absorbance value of cy3 at 550 nm.
Although the invention has been described and illustrated in some detail, it should be understood that various modifications may be made to the described embodiments or equivalents may be substituted, as will be apparent to those skilled in the art, without departing from the spirit of the invention.
Claims (6)
1. A method for preparing a multi-fluorescent nucleic acid probe, which is characterized by comprising the following steps:
A. carrying out nucleic acid amplification by using 5-ethynyl-uracil deoxynucleotide (5-EdUTP) to replace thymine deoxynucleotide (dTTP) to obtain double-stranded DNA of high-density alkyne;
B. treating the double-stranded DNA obtained in the step A by Exonuclease Lambda Exonuclease, and digesting the nucleic acid chain of the 5' end phosphorylation marker to obtain high-density alkyne single-stranded DNA, namely a probe chain;
C. through a copper ion catalyzed click reaction, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne to obtain the multi-fluorescent nucleic acid probe.
2. The method of claim 1, wherein the first fluorescent nucleic acid probe is a fluorescent probe,
the PCR reaction system of the nucleic acid amplification in the step A is as follows: 1 XPCR Buffer (Mg2+ Plus), 1. mu.M forward and 1. mu.M downstream primers, 20 pg/100. mu.L Template, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM EdUTP, 2.5 units/100. mu.L TaKaRa Taq enzyme, the remainder being ddH2O；
Carrying out PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: at 95 ℃ for 30 s; 30s at 55 ℃; at 72 ℃ for 30s, and after 40 cycles, at 72 ℃ for 5 min; and purifying the amplification product by using a kit to obtain pure double-stranded DNA.
3. The method for preparing the multi-fluorescent nucleic acid probe according to claim 1, wherein the double-stranded DNA and the Lambda Exonuclease Exonuclease are mixed according to a ratio of 2 μ g/10U in the step B, and the incubation conditions are as follows: 30min at 37 ℃; 75 ℃ for 10 min.
4. The method of claim 1, wherein the conditions for the click reaction in step C are 60. mu.L of single-stranded DNA solution, 20. mu.L of 100mM cy3-azide, 10. mu.L of 0.1M PBS buffer, pH7.4, and 10. mu.L of CuAAC catalytic solution, and incubation at 37 ℃ for 60 min.
5. The method for preparing the multi-fluorescent nucleic acid probe according to claim 4, wherein the CuAAC catalytic solution is prepared by: 70 mu L H2O，4μL 100mM THPTA，1μL100mM CuSO425 μ L of 100mM sodium ascorbate solution was mixed well and left at room temperature for 20 min.
6. The use of a multi-fluorescent nucleic acid probe according to claim 1, wherein said multi-fluorescent nucleic acid probe is used in a biosensor.
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| CN112552363A (en) * | 2020-12-24 | 2021-03-26 | 山东大学 | Ascorbic acid coupled nucleotide probe and preparation method and application thereof |
Citations (2)
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|---|---|---|---|---|
| CN104059907A (en) * | 2013-03-13 | 2014-09-24 | 安捷伦科技有限公司 | Dendrimeric Dye-containing Oligonucleotide Probes And Methods Of Preparation And Uses Thereof |
| CN104792753A (en) * | 2015-04-07 | 2015-07-22 | 上海大学 | Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction |
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Patent Citations (2)
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| CN104059907A (en) * | 2013-03-13 | 2014-09-24 | 安捷伦科技有限公司 | Dendrimeric Dye-containing Oligonucleotide Probes And Methods Of Preparation And Uses Thereof |
| CN104792753A (en) * | 2015-04-07 | 2015-07-22 | 上海大学 | Fluorescent biosensing method used for detecting low molecular ligand target protein and based on combination of inhibition of click chemistry reaction |
Non-Patent Citations (1)
| Title |
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| FABIAN TOLLE等: "A Versatile Approach Towards Nucleobase-Modified Aptamers", ANGEW CHEM INT ED ENGL, vol. 54, no. 37, pages 1, XP055589969, DOI: 10.1002/anie.201503652 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552363A (en) * | 2020-12-24 | 2021-03-26 | 山东大学 | Ascorbic acid coupled nucleotide probe and preparation method and application thereof |
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| CN113150769B (en) | 2023-12-22 |
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