CN113150769B - Preparation method and application of multi-fluorescent nucleic acid probe - Google Patents
Preparation method and application of multi-fluorescent nucleic acid probe Download PDFInfo
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/14—Macromolecular compounds
- 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-fluorescent 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; after the obtained double-stranded DNA is treated by exonuclease Lambda Exonuclease, 5' -end phosphorylation labeled nucleic acid chains are digested, so that high-density alkyne single-stranded DNA is obtained; through a click reaction catalyzed by copper ions, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne, and the multi-fluorescence nucleic acid probe is obtained. The fluorescent groups of the multi-fluorescent nucleic acid probe prepared by the invention are increased, so that signal amplification is realized structurally, the sensitivity of the fluorescent nucleic acid probe in researches such as analysis detection and biological imaging can be enhanced, and the practical value of the fluorescent 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-fluorescent nucleic acid probe.
Background
A nucleic acid probe is a nucleic acid fragment with a 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 are changed when the molecule with specific interaction with the nucleic acid probe exists, so that the signal change is caused, and the nucleic acid probe can be used for target molecule identification in the fields of chemistry, biology, medicine, pharmacy and the like. Among the numerous nucleic acid probes, fluorescent nucleic acid probes are one of the research hotspots of analytical chemistry because of their high sensitivity, good specificity, design diversity, and strong quantitative analysis capability. The fluorescent nucleic acid probe mainly comprises a target recognition unit and a signal transduction unit. The functional nucleic acid is one of ideal recognition units of fluorescent nucleic acid probes, consists of a segment of nucleic acid fragments with known sequences, and has the advantages of good stability, strong binding force, good biocompatibility, easiness in synthesis and modification and the like. The fluorescent reporter unit is typically an organic dye molecule or an inorganic fluorescent nanomaterial that is responsible for converting the change in chemical environment caused by 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, nucleic acid aptamer probes and the like, and the signal output of the molecular beacon type nucleic acid probes, the strand displacement type nucleic acid probes and the nucleic acid aptamer probes depend on a single fluorescent group at the tail end of the probe, namely the ratio of the probe to the fluorescent group is 1:1, which limits the sensitivity of the probe to some extent, affects 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 in method, easy to manufacture and suitable for common laboratories, and the fluorescent groups of the multi-fluorescent nucleic acid probe synthesized by the method are increased, so that signal amplification is realized structurally, the sensitivity of the fluorescent nucleic acid probe in researches such as analysis detection and biological imaging is enhanced, and the practical value of the fluorescent nucleic acid probe is improved.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for preparing a multi-fluorescent nucleic acid probe, comprising the steps of:
A. performing 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. the double-stranded DNA obtained in the step A is digested by 5' -end phosphorylation labeled nucleic acid strand after being treated by exonuclease Lambda Exonuclease, so as to obtain high-density alkyne single-stranded DNA, namely a probe strand;
C. through a click reaction catalyzed by copper ions, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne, and the multi-fluorescence nucleic acid probe is obtained.
Further, the PCR reaction system for amplifying the nucleic acid in the step A is as follows: 1 XPCR Buffer (Mg2+Plus), 1. Mu.M upstream primer and 1. Mu.M downstream primer, 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 balance ddH 2 O; performing PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: 95 ℃ for 30s;55 ℃ for 30s;72 ℃,30s, 40 cycles, 72 ℃ and 5min; the amplified product is purified by the kit to obtain the pure double-stranded DNA.
Further, the double-stranded DNA and Lambda Exonuclease exonuclease in the step B are mixed according to the proportion of 2 mug/10U, and the incubation conditions are as follows: 37 ℃ for 30min;75 ℃ for 10min.
Further, the conditions for the ligation reaction in step C were 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, 10. Mu.L of CuAAC catalyst, and incubation at 37℃for 60min. Wherein the preparation of the CuAAC catalytic liquid comprises the following steps: 70 mu L H 2 O，4μL 100mM THPTA，1μL 100mM CuSO 4 mu.L of 100mM sodium ascorbate solution was thoroughly mixed and left at room temperature for 20min.
The invention also provides application of the multi-fluorescence nucleic acid probe, which is used in a biosensor, such as a biosensor of graphene-nucleic acid probe, wherein the multi-fluorescence nucleic acid probe is adsorbed on the surface of the graphene nano-sheet, and fluorescence resonance energy transfer occurs between fluorescent molecules and the nano-sheet, so that fluorescence quenching is realized; when the detected target nucleic acid is combined with the probe to form double chains, namely, the double chains are desorbed from the surface of the graphene, fluorescence resonance energy transfer disappears, fluorescence is recovered, and a fluorescence process is realized from nothing to nothing. According to the fact that the recovery intensity of fluorescence is proportional to the concentration of the target, the target is quantitatively analyzed, and the sensitivity of the target is improved by orders of magnitude compared with that of a 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, the synthesis steps are few, the required time is short, and the multi-fluorescent nucleic acid probe can be quickly synthesized in a common laboratory.
(3) The multi-fluorescent nucleic acid probe prepared by the invention can be widely applied to various fields such as biosensors, biological imaging and the like.
Drawings
FIG. 1 is an agarose gel electrophoresis diagram of a probe synthesis process;
FIG. 2 is a schematic diagram of ultraviolet absorbance spectra of synthesized probe strands and standard probes.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 preparation of Multi-fluorescent nucleic acid probes for detection of EV-71 Virus
A. Preparation of double-stranded DNA:
the template sequence, the upstream primer and the downstream primer are synthesized by Takara biological company.
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';
an upstream primer: 5'-GAG CAG TCA CAG TCC AGA AG-3' (5 ' -terminal phosphorylation)
A downstream primer: 5'-GTA TCG TGC AAG GGT GAA TGC-3'
PCR reaction system
Performing PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: 95 ℃ for 30s;55 ℃ for 30s;72 ℃,30s, 40 cycles, 72 ℃ and 5min. The amplified product is purified by the kit to obtain the pure double-stranded DNA.
B. Preparation of a probe single strand:
the double-stranded DNA obtained in the step A was mixed with Lambda Exonuclease exonuclease in a ratio of 2. Mu.g/10U under the following incubation conditions: 37 ℃ for 30min;75 ℃ for 10min. Digesting the 5' -end phosphorylated labeled nucleic acid chain, and purifying to obtain high-density alkyne single-stranded DNA;
the probe strand sequence is: 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:
preparation of CuAAC catalytic liquid: 70 mu L H, 4 mu L THPTA (100 mM), 1 mu L CuSO 4 (100 mM), 25. Mu.L of sodium ascorbate solution (100 mM) was thoroughly mixed and left at room temperature for 20min; 60. Mu.L of single-stranded DNA solution, 20. Mu.L of cy3-azide (100 mM), 10. Mu.L of PBS (0.1M, pH 7.4), 10. Mu.L of LCuAAC catalyst solution, and incubation at 37℃for 60min. Purifying by the kit to obtain the simple multi-fluorescent nucleic acid probe.
D. Adsorbing the multi-fluorescent nucleic acid probe on the surface of the graphene nano-sheet to prepare the biosensor of the graphene-nucleic acid probe. Fluorescence quenching due to fluorescence resonance energy transfer between fluorescent molecules and the nanosheets; when the detected target nucleic acid is combined with the probe to form double chains, namely, the double chains are desorbed from the surface of the graphene, fluorescence resonance energy transfer disappears, fluorescence is recovered, and a fluorescence process is realized from nothing to nothing. According to the fact that the recovery intensity of fluorescence is proportional to the concentration of the target, the target is quantitatively analyzed, and the sensitivity of the target is improved by orders of magnitude compared with that of a traditional fluorescent probe.
EXAMPLE 2 comparison of EdUTP with double-stranded DNA synthesized by dTTP
Double-stranded DNA was obtained by replacing EdUTP with dTTP as in step A of example 1. 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 strand were analyzed by agarose gel electrophoresis. The method comprises the following steps:
and (3) glue preparation: adding accurately weighed agarose powder into a conical flask containing 60mL of 1 XTBE buffer, heating and dissolving in a microwave oven, cooling to about 50-60 ℃, adding ethidium bromide, fully mixing, introducing into a gel-making mold, and solidifying the gel at room temperature.
Loading: a proper amount of sample was mixed with 6 Xloading buffer, and the loading amount of each sample tank was 10. Mu.L.
Electrophoresis: after the sample is added, the electrophoresis tank cover is closed, and the power supply is immediately connected. The control voltage was kept at 90V, and when the strip was moved to about 2cm from the front of the gel, electrophoresis was stopped, photographed under ultraviolet rays, and observed, resulting in fig. 1.
FIG. 1 is an agarose gel electrophoresis of each DNA strand of step A, B, 1 being a double strand synthesized with A, G, C, T four nucleotides; 2 is high density alkyne double stranded DNA synthesized with EdUTP; 3 is purified high-density alkyne double-stranded DNA;4 is high-density alkyne single-stranded DNA obtained after exonuclease digestion, namely a probe chain. The result of agarose gel electrophoresis 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-stranded probe.
EXAMPLE 3 comparison of synthetic Probe Strand 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',5' end is connected with a fluorescent molecule;
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 has a plurality of fluorescent molecules attached thereto;
when the concentration of the single-stranded DNA was uniform by diluting both probes to 32 ng/. Mu.L, the absorbance of cy3 at 550nm was measured by Nanodrop 2000, and the results are shown in FIG. 2.
The ultraviolet absorption spectrum shown in FIG. 2 is a schematic diagram, and the absorbance value of cy3 at 550nm is compared, so that the fluorescent molecule of the multi-fluorescent nucleic acid probe synthesized by the invention is 4.6 times that of a purchased standard probe.
While the invention has been described and illustrated in considerable detail, it should be understood that modifications and equivalents to the above-described embodiments will become apparent to those skilled in the art, and that such modifications and improvements may be made without departing from the spirit of the invention.
Claims (6)
1. A method for preparing a multi-fluorescent nucleic acid probe, comprising the steps of:
A. performing 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. the double-stranded DNA obtained in the step A is digested by 5' -end phosphorylation labeled nucleic acid strand after being treated by exonuclease Lambda Exonuclease, so as to obtain high-density alkyne single-stranded DNA, namely a probe strand;
C. through a click reaction catalyzed by copper ions, the dye cy3-azide with azido is successfully modified on single-stranded DNA of high-density alkyne, and the multi-fluorescence nucleic acid probe is obtained.
2. The method for preparing a multi-fluorescent nucleic acid probe according to claim 1, wherein,
the PCR reaction system for amplifying the nucleic acid in the step A is as follows: 1 XPCR Buffer (Mg) 2+ Plus), 1. Mu.M upstream primer and 1. Mu.M downstream primer, 20 pg/100. Mu.L Template,0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM EdUTP, 2.5 units/100. Mu.LTaKaRaTaq enzyme, the balance ddH 2 O；
Performing PCR reaction according to the PCR reaction system, wherein the amplification conditions are as follows: 95. 30℃, s; 55. 30℃, s; 72. 30. 30s, after 40 cycles, 72℃for 5min; the amplified product is purified by the kit to obtain the pure double-stranded DNA.
3. The method for preparing a multi-fluorescent nucleic acid probe according to claim 1, wherein the double-stranded DNA and Lambda Exonuclease exonuclease are mixed in the ratio of 2 μg/10U in the step B, and the incubation conditions are as follows: 37. at the temperature of 30min; 75. at a temperature of 10min.
4. The method for preparing a multi-fluorescent nucleic acid probe according to claim 1, wherein the condition of the clicking reaction in the step C is 60. Mu.L of single-stranded DNA solution, 20. Mu.L of 100mM cy3-azide, 10. Mu.L of 0.1 MPBS buffer pH7.4, 10. Mu.L of UAAC catalyst, and incubation at 37℃for 60min.
5. The method for preparing a multi-fluorescent nucleic acid probe according to claim 4, wherein the CuAAC catalyst solution is prepared by: 70. mu LH 2 O，4 μL100 mM THPTA，1μL 100mM CuSO 4 mu.L of 100mM sodium ascorbate solution was thoroughly mixed and left at room temperature for 20min.
6. The use of a multi-fluorescent nucleic acid probe according to claim 1, wherein the multi-fluorescent nucleic acid probe is used in a biosensor; the application is for non-diagnostic purposes.
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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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