CN114672483B - Method for preparing nucleic acid probe by ultrasonic method - Google Patents
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Abstract
The invention provides a method for preparing a nucleic acid probe by an ultrasonic method, wherein the nucleic acid probe is prepared by carrying out ultrasonic reaction on nucleic acid with an amino active group and an activated ester modified dye capable of reacting with the amino group in an alkaline buffer solution at the temperature of 30-50 ℃. The reaction can be completed in only 5min in the production process, so that the reaction rate is greatly improved, and the reaction period is greatly shortened; meanwhile, the invention greatly improves the utilization rate of the raw materials of the modified dye.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for preparing a nucleic acid probe by an ultrasonic method.
Background
Single-stranded DNA and RNA are composed of a certain number of deoxynucleotides or combinations of nucleotides. And the DNA modified probe and the RNA modified probe are formed by modifying the structure of the DNA or the RNA. The DNA modified probe and the RNA modified probe are widely applied to the fields of molecular diagnosis QPCR (real-time fluorescence quantification), STR (short tandem repeat), FISH (fluorescence in situ hybridization) and the like.
Generally, there are two common methods available for the engineering of single stranded DNA and RNA. One is the solid phase phosphoramidite triester method and the other is the liquid phase amino activated ester addition method. The traditional liquid phase amino-activated ester addition method is that DNA or RNA containing amino active groups is dissolved in alkaline buffer solution, then 3 times of molar amount of activated ester modified dye dissolved in organic solvent is added, reaction is carried out for 4-12 hours at normal temperature, then alcohol is added for precipitation, meanwhile, the precipitate is repeatedly washed by alcohol, precipitated solid is taken out, then water is used for dissolution, and finally, the pure DNA modified probe or RNA modified probe is obtained after purification by a high performance liquid chromatograph.
The liquid phase amino-activated ester addition method requires the synthesis of an amino structure (NH 2) on DNA or RNA, while requiring the activated ester modification dye to be a succinimide-activated ester structure, as shown in the following reaction scheme (1), which is easier to synthesize than phosphoramidite structures.
The specific operating scheme of the liquid phase amino activated ester addition process is as follows:
(1) dissolving 100nmol DNA/RNA containing amino structure in 100ul 0.5MNa with pH 8.5 2 CO 3 /NaHCO 3 In the buffer solution, shaking fully and mixing uniformly.
(2) Dissolving 300nmol of succinimide activated ester modified dye solid in 37.5ul of DMF solvent, adding into the buffer solution, and shaking and mixing at room temperature.
(3) Placing the solution into a shaking table, and shaking for 4-12 hours.
(4) Adding 2mL of alcohol into the above solution, freezing at-20 deg.C for 30 min to obtain flocculent precipitate, centrifuging at high speed of 12000 rpm, removing supernatant, and collecting precipitate.
(5) Washing the precipitate with 2 ml-20 deg.c alcohol solution for 3 times, and stoving the precipitate to obtain coarse DNA/RNA modified probe product.
(6) And dissolving the dried DNA/RNA modified probe crude product by using 1mL of ultrapure water, loading the dissolved DNA/RNA modified probe crude product on a high performance liquid chromatograph for purification, and taking a pure product.
The liquid phase amino activated ester addition process has the following disadvantages:
(1) because DNA/RNA is easily soluble in water, and most of the solid succinimide-activated ester modified dye is insoluble in water, the solid succinimide-activated ester modified dye needs an organic solvent for dissolution. In order to make the reaction system favor homogeneous reaction, organic solvents with high water-solubility are required, and the organic solvents have high toxicity.
(2) Since organic solvents and buffer salts are used in the reaction process, which must be removed in advance in the subsequent production, ethanol is used in a large amount in the post-reaction treatment for post-precipitation washing. Resulting in more organic solvent waste.
(3) The molar ratio of the succinimide-activated ester-modified dye to the DNA/RNA substrate is about 3:1, but since the reaction is aqueous, although the succinimide-activated ester-modified dye reacts preferentially with DNA/RNA having an amino structure, the succinimide-activated ester-modified dye also reacts with water relatively quickly, and the final molar yield of the liquid phase amino-activated ester addition process is still not high, mostly between 20-30%, i.e., only 7-10% in terms of the modified dye utilization. The material costs are still too high.
(4) Because the internal structure of DNA/RNA is complex to wrap, when the DNA contains more than 50 deoxynucleotides and the RNA contains more than 40 nucleotides, the amino structure carried by the DNA/RNA is difficult to contact and react with the succinimide activated ester modified dye, and the yield of the traditional liquid-phase amino activated ester addition method is extremely low.
The synthesis process of the solid phase phosphoramidite triester method mainly comprises 4 steps of deprotection, coupling, capping and oxidation. Every 4 steps are completed to connect one deoxynucleotide or nucleotide, and by repeating the 4 steps, the deoxynucleotides or nucleotides are connected to form DNA or RNA.
The solid phase phosphoramidite triester method can also be used to synthesize a modified dye onto a DNA or RNA strand during the synthesis of DNA/RNA.
Solid phase phosphoramidite triester process
(1) The solid phase phosphoramidite triester method is a solid-liquid two-phase reaction, the ratio of the required modified dye to the DNA/RNA substrate is more than 20:1, the molar yield is about 30-40%, namely the utilization rate of the modified dye is only 1.5-2%, and the material cost is wasted greatly.
(2) The reaction process is chemically synthesized by a four-step method, a large amount of organic solvent is used, and harmful waste liquid is generated.
(3) A modified probe is synthesized by utilizing a solid-phase phosphoramidite triester method, and a modified dye solid with an active chemical structure of a phosphoramidite monomer structure is needed, namely a reaction formula compound (8). For some modified probes with complicated structures (such as CY5, ROX, etc.), it is difficult to synthesize active phosphoramidite monomers, or the subsequent process is limited, which limits the application of solid phase phosphoramidite triester method to synthesize modified probes.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for preparing a nucleic acid probe by an ultrasonic method, wherein nucleic acid with an amino active group and an active ester modified dye capable of reacting with the amino group are subjected to an ultrasonic reaction in an alkaline buffer solution at the temperature of 30-50 ℃ to prepare the nucleic acid probe, and the frequency of the ultrasonic reaction is 35-50 KHZ.
In one embodiment, the nucleic acid probe is prepared by ultrasonic reaction of nucleic acid with amino active group and active ester modified dye capable of reacting with amino group in alkaline buffer solution at 45 ℃.
In one embodiment, the nucleic acid is DNA or/and RNA and the activated ester modification dye is a succinimide-activated ester or isothiocyanate-activated ester modification dye.
In one embodiment, the time of the ultrasonic reaction is 5 to 15 minutes.
In one embodiment, the time of the ultrasonic reaction is 5 to 10 minutes.
In one embodiment, the ultrasonic reaction has a frequency of 40 KHZ.
In one embodiment, the ratio of the nucleic acid having an amino reactive group to the activated ester-modifying dye that is reactive with an amino group is from 1:1 to 1: 4.
In one embodiment, the ratio of the nucleic acid having an amino reactive group to the activated ester-modifying dye that is reactive with an amino group is 1: 2.
In one embodiment, the alkaline buffer solution has a pH of 7.1 to 10.0.
In one embodiment, the alkaline buffer solution is Na at pH 8.5 2 CO 3 /NaHCO 3 And (4) a buffer solution.
In one embodiment, after the ultrasonic reaction, adding alcohol to the buffer solution for precipitation, then centrifuging, removing the supernatant, and taking the precipitate; washing the precipitate with alcohol to obtain the modified nucleic acid probe crude product.
In one embodiment, the crude nucleic acid probe is dissolved in ultrapure water, loaded onto a high performance liquid chromatograph, and purified to obtain a pure nucleic acid probe.
In one embodiment, the number of bases in the nucleic acid is not less than 22.
In one embodiment, the number of bases in the nucleic acid is not less than 46.
The DNA/RNA having amino groups of the present invention refers to all of the DNAs/RNAs having amino groups at the 3-terminal, 5-terminal and intermediate positions.
The activated ester of the modified dye of the invention includes, but is not limited to, CY5.5, SE (Cyanine 5.5 succinimide activated ester), HEX, SE (hexachlorofluorescein succinimide activated ester), CY3 (Cyanine 3 succinimide activated ester), JOE, SE (4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimide activated ester), TET, SE (tetrachlorofluorescein succinimide activated ester), TAMRA, SE (hydroxy tetramethyl rhodamine succinimide activated ester), ROX, SE (6-hydroxy-X-rhodamine succinimide activated ester), FAM, SE (fluorescein succinimide activated ester), FITC (fluorescein isothiocyanate activated ester), Texas Red, SE (Texas Red succinimide activated ester), CY5 (Cyanine 5 succinimide activated ester), DIG (digoxigenin succinimide activated ester), Dylight dye SE series (Dylight series dyes), Alexa dye SE series (Alexa dye series), AMCA-X SE, ATTO dye SE series (ATTO dye series).
The liquid phase amino-activated ester ultrasonic addition method is a method for quickly and efficiently synthesizing a DNA/RNA modified probe. Dissolving single-stranded nucleic acid with an amino active group in an alkaline buffer solution, adding 1-4 times of the molar weight of modified dye activated ester dissolved in an organic solvent, placing the mixture in an ultrasonic instrument, heating and ultrasonically treating the mixture for 5-15 minutes at the temperature of 30-50 ℃, and promoting the accelerated completion of the reaction by utilizing the cavitation effect generated by ultrasonic waves; and then adding alcohol for precipitation, simultaneously washing the precipitate repeatedly by using the alcohol, taking the precipitate solid, adding ultrapure water, rapidly dissolving by using ultrasound, and directly loading the precipitate solid on a high performance liquid chromatograph for purification to obtain a pure DNA modified probe or RNA modified probe. The invention does not need to wait for 4-12 hours of reaction in the production process, and can finish the reaction only in 5min, thereby greatly improving the reaction rate and greatly shortening the reaction period; meanwhile, the invention greatly improves the utilization rate of the modified dye, particularly for long-chain nucleic acid, such as nucleic acid with more than 46 bases.
More importantly, the invention solves the problem of limited application of the solid phase phosphoramidite triester method and the problem of difficult production of the DNA/RNA modified probe with a complex structure by the traditional liquid phase amino-activated ester addition method.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. 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 application. In the following examples, unless otherwise specified, all methods are conventional in the art.
Example a basic principle of the method of the invention
Cavitation is generated by ultrasonic waves, so that DNA/RNA with an amino structure is quickly combined with the succinimide activated ester modified dye, and the chemical reaction rate is improved; meanwhile, because the reaction time is extremely short, the succinimide activated ester modified dye cannot be degraded, and side reactions are greatly reduced, so that the reaction can be fully carried out. The reaction chemical equation of the invention is as follows:
specific embodiments are as follows:
the method comprises the following steps: 200nmol DNA/RNA containing amino structure was dissolved in 200ul 0.5M Na pH 8.5 2 CO 3 /NaHCO 3 Fully shaking and uniformly mixing in a buffer solution;
step two: dissolving 600nmol of succinimide activated ester modified dye in 75ul of DMF solvent, adding the solution into the buffer solution in the step one, and shaking and mixing uniformly;
step three: putting the solution into an ultrasonic instrument, and heating and carrying out ultrasonic treatment at 45 ℃ for 5-10 minutes;
step four: adding alcohol into the solution for precipitation, centrifuging at high speed of 12000 r, removing supernatant, and collecting precipitate;
step five: washing the precipitate with alcohol for 3 times, and drying the precipitate to obtain a crude product of the DNA/RNA modified probe;
step six: and dissolving the dried DNA/RNA modified probe crude product by using ultrapure water, loading the dissolved product onto a high performance liquid chromatograph, purifying by using 0.1M triethylamine acetate and acetonitrile, and taking a pure product.
The principle of the invention is as follows:
1. the cavitation phenomenon is generated by ultrasonic waves, namely, micro bubbles existing in a reaction system are activated under the action of an ultrasonic field to generate a very transient high-energy environment, so that a DNA/RNA chain with a complex structure is effectively unfolded, more groups with amino structures are exposed, and the reaction effect is improved.
2. The reaction is carried out in homogeneous solution, bubbles formed in the ultrasonic cavitation process not only contain vapor generated by liquid, but also contain gas dissolved in the liquid, and when the cavitation bubbles collapse, the generated energy can cause bond breakage, promote the generation of free radicals, change the solvent structure and accelerate the reaction speed.
EXAMPLE two comparison experiments of different ultrasonic frequencies of DNA/RNA with amino active group and succinimide activated ester modified dye
1. The invention screens the frequency of ultrasonic waves:
a. 300nmol of DNA/RNA containing amino structures was dissolved in 300ul of 0.5M Na pH 8.5 2 CO 3 /NaHCO 3 In a buffer solution;
b. dissolving 3600nmol of succinimide activated ester modified dye by using 450ul of DMF solvent, fully shaking and uniformly mixing;
c. adding 112.5ul of the activated ester solution into the DNA/RNA solution (a), and carrying out vortex oscillation for 5 seconds to form a solution with the molar ratio of the DNA/RNA substrate to the activated ester being 1: 3;
d. dividing the solution into 3 parts, and reacting at room temperature for 10 minutes at three different ultrasonic frequencies of 28KHz, 40KHz and 60 KHz;
e. and (3) performing alcohol precipitation post-treatment, performing HPLC purification by adopting 0.1M triethylamine acetate and acetonitrile, calculating the molar yield, and comparing, wherein the base number in the table refers to the number of deoxynucleotides or nucleotides in DNA/RNA. Specific data are shown in table 1, and the sequence of each sample is shown in table 2:
TABLE 1
TABLE 2
And (4) conclusion: since the intensity of the cavitation effect is directly related to the frequency, the higher the frequency, the smaller the cavitation bubbles, the weaker the cavitation intensity, and the greater the degree of attenuation thereof. From data, the ultrasonic frequency of 40KHZ is better than that of 60K HZ in the effect of promoting reaction efficiency, and under the ultrasonic frequency of 28KHZ, the cavitation effect is too strong, which directly causes the breakage of the chemical bonds of the substrate skeleton of the modified dye, and the reaction is finally very complicated, so that a pure product is difficult to obtain.
On the basis, the inventor further verifies the influence of different frequencies on molar yield on the basis of the ultrasonic frequency of 40KHZ under the condition of keeping the ultrasonic reaction conditions unchanged, the inventor selects to test 30KHZ, 35KHZ, 38KHZ, 40KHZ, 42KHZ, 45KHZ, 50KHZ and 55KHZ at room temperature and reaction time of 10 minutes, and samples are DNA-ROX-1 and RNA-CY5-1, compared with 40 KHZ:
a. 30KHZ DNA-ROX-1 and RNA-CY5-1 molar yields were 37% and 54% of the average molar yield of 40KHZ, respectively;
b. 35KHZ of DNA-ROX-1 and RNA-CY5-1 molar yields were 67% and 76% of the average molar yield of 40KHZ, respectively;
c. 38KHZ DNA-ROX-1 and RNA-CY5-1 molar yields were 87% and 90% of the average molar yield of 40KHZ, respectively;
d. the DNA-ROX-1 and RNA-CY5-1 molar yields of 42KHZ were 106% and 111% of the average molar yield of 40KHZ, respectively;
e. the DNA-ROX-1 and RNA-CY5-1 molar yields of 45KHZ were 96% and 105% of the average molar yield of 40KHZ, respectively;
f. the 50KHZ DNA-ROX-1 and RNA-CY5-1 molar yields were 82% and 96% of the 40KHZ average molar yield, respectively;
g. the 55KHZ DNA-ROX-1 and RNA-CY5-1 molar yields were 45% and 68% of the 40KHZ average molar yield, respectively.
According to the above experimental results, it is preferable to select 35KHZ to 50 KHZ.
EXAMPLE III comparison of the duration of sonication of DNA/RNA with amino reactive groups and of the dye modified with succinimide-activated ester
a. 400nmol DNA/RNA containing amino structures was dissolved in 400ul 0.5M Na pH 8.5 2 CO 3 /NaHCO 3 In a buffer solution;
b. dissolving 4800nmol of succinimide activated ester modified dye in 600ul of DMF solvent, and shaking and mixing uniformly;
c. adding 150ul of the activated ester solution (b) into the DNA/RNA solution (a), and performing vortex oscillation for 5 seconds to form a solution with the molar ratio of the DNA/RNA substrate to the activated ester being 1: 3;
d. dividing the solution of the step (c) into four parts, and reacting at 40KHZ ultrasonic frequency and room temperature for 2min, 5min, 10min and 15min respectively;
e. and (3) performing alcohol precipitation post-treatment, performing HPLC purification by adopting 0.1M triethylamine acetate and acetonitrile, calculating the molar yield, and comparing, wherein the base number in the table refers to the number of deoxynucleotides or nucleotides in DNA/RNA. Specific data are shown in table 3:
TABLE 3
And (4) conclusion: from the data, the maximum reaction yield is achieved after the primer with the shorter base number is subjected to ultrasonic reaction for 5min, the maximum reaction yield is achieved after the primer with the slightly longer base number is subjected to ultrasonic reaction for 10min, and the reaction efficiency of 15min is similar to that of 5 min.
EXAMPLE IV comparison of different ultrasound temperatures for DNA/RNA with amino reactive groups and for succinimidyl-activated ester-modified dyes
a. 100nmol DNA/RNA containing amino structure was dissolved in 100ul 0.5M Na pH 8.5 2 CO 3 /NaHCO 3 In a buffer solution;
b. dissolving 4800nmol of succinimide activated ester modified dye in 600ul of DMF solvent, and fully shaking and mixing uniformly;
c. adding 37.5ul of the activated ester solution (b) into the DNA/RNA solution (a), and performing vortex oscillation for 5 seconds to form a solution with the molar ratio of the DNA/RNA substrate to the activated ester being 1: 3;
d. reacting for 10 minutes at different temperatures of 25-60 ℃ under the ultrasonic frequency of 40 KHZ;
e. and (3) performing alcohol precipitation post-treatment, performing HPLC purification by adopting 0.1M triethylamine acetate and acetonitrile, calculating the molar yield, and comparing, wherein the base number in the table refers to the number of deoxynucleotides or nucleotides in DNA/RNA. Specific data are shown in table 4 and sample sequences are shown in table 5:
TABLE 4
TABLE 5
And (4) conclusion: from data, the heating temperature of the ultrasonic reaction is optimal at 45 ℃, and when the temperature is too low, the reaction is not sufficiently performed; when the temperature is too high, the modified primer may be partially degraded, and the reaction is finally very complicated, resulting in a decrease in yield.
EXAMPLE V comparison of the molar ratios of DNA/RNA with amino-reactive groups and of the modified dye with activated ester of succinimide
Comparison of the molar amounts of DNA/RNA substrate and modified dye
a. 100nmol DNA/RNA containing amino structure was dissolved in 100ul 0.5M Na pH 8.5 2 CO 3 /NaHCO 3 In a buffer solution;
b. dissolving 4000nmol of succinimide activated ester modified dye in 500ul of DMF solvent, fully shaking and mixing uniformly;
c. adding the activated ester solution (b) with different volumes into the DNA/RNA solution (a) according to a set molar ratio, and carrying out vortex oscillation for 5 seconds to form solutions with different molar concentration ratios;
d. reacting for 10 minutes at 45 ℃ under 40KHZ ultrasonic frequency;
e. and (3) performing alcohol precipitation post-treatment, performing HPLC purification by adopting 0.1M triethylamine acetate and acetonitrile, and comparing the molar yield of the DNA/RNA, wherein the base number in the table refers to the number of deoxynucleotides or nucleotides in the DNA/RNA. The experimental data are shown in table 6:
TABLE 6
And (4) conclusion: through substrate concentration screening, the mole ratio of DNA/RNA with an amino structure to the succinimide-activated ester modified dye is judged to be the optimal ratio of 1:2, and the succinimide-activated ester modified dye is added on the basis of the mole ratio of 1:2, so that the yield is not greatly influenced; the cost of the dye is high in probe synthesis, and the amount of the dye used is reduced as much as possible in order to save cost.
2. A liquid-phase amino activated ester ultrasonic addition experiment is carried out according to the molar ratio of DNA/RNA to an activated ester modified dye of 1:2, the ultrasonic frequency of 40KHz, the temperature of 45 ℃ and the reaction time of 10 minutes, and a traditional liquid-phase amino activated ester addition experiment in which the molar ratio of DNA/RNA to an activated ester is 1:3 is compared in parallel, wherein the specific experimental conditions of the traditional liquid-phase amino activated ester addition experiment are completed according to the experimental conditions explained in the background art of the invention. And (3) calculating the molar yield of the two methods and comparing the molar yield with the utilization rate of the modified dye, wherein the base number in the table refers to the number of deoxynucleotides or nucleotides in the DNA/RNA. The comparative data are shown in Table 7:
TABLE 7
And (4) conclusion: it can be seen that the liquid phase amino-activated ester ultrasonic addition method greatly improves the yield no matter DNA or RNA; particularly, in longer nucleic acid, the liquid-phase amino-activated ester ultrasonic addition method has more advantages than the traditional liquid-phase amino-activated ester addition method, and provides a new reliable process for large-scale industrial production.
The results of the liquid phase amino activated ester ultrasonic addition/conventional liquid phase amino activated ester addition comparison are shown in table 8 below and the corresponding sample sequences are shown in table 9.
TABLE 8
TABLE 9
From the above, it can be seen that, for nucleic acid, the product molar yield and the utilization rate of unit modified dye of the liquid-phase amino-activated ester ultrasonic addition method of the invention are significantly increased compared with the traditional liquid-phase amino-activated ester addition method, especially for RNA or DNA with the length of more than 46 bases, the advantages of the method of the invention are more obvious, and the method of the invention can effectively solve the technical problem of probe modification of long nucleic acid.
3. In order to verify the promotion condition of the invention on the synthesis efficiency of the DNA/RNA modified probe with a complex structure, a liquid-phase amino activated ester ultrasonic addition experiment is carried out according to the reaction conditions that the mole ratio of DNA/RNA and activated ester is 1:2, the ultrasonic frequency is 40KHZ, the reaction time is 10min, and the reaction temperature is 45 ℃, and the traditional liquid-phase amino activated ester addition experiment with the ratio of DNA/RNA and activated ester being 1:3 is compared in parallel, wherein the specific experimental conditions of the traditional liquid-phase amino activated ester addition experiment are completed according to the experimental conditions explained in the background technology of the invention. The molar yields of the two methods were calculated and compared, and the specific data are shown in table 10:
watch 10
And (4) conclusion: the data show that the molar yield of the ultrasonic method for the synthesis efficiency of the DNA/RNA modified probe with a complex structure is obviously higher than that of the traditional activated ester addition method, and the ester connecting efficiency of the DNA/RNA modified probe with a complex structure is remarkably improved.
EXAMPLE VI DNA/RNA with amino reactive groups and ultrasonic reaction experiments with isothiocyanate-activated ester modified dyes
In order to expand the general applicability of the experiment, the patent simultaneously researches liquid-phase amino-activated ester ultrasonic addition experiments of DNA and RNA and isothiocyanate-activated ester modified dye with different molar ratios, and finds that the invention also has good reaction effect on the isothiocyanate-modified dye, the reaction principle is shown as the following reaction formula, and the specific data are shown as the following table 11 and the sample sequence is shown as the following table 12:
the specific operation scheme is as follows:
a. dissolving 100nmol DNA/RNA containing amino structure in 100ul 0.5M Na with pH of 8.5 2 CO 3 /NaHCO 3 In a buffer solution
b. Dissolving 4000nmol of isothiocyanate activated ester modified dye in 500ul of DMF solvent, fully shaking and uniformly mixing;
c. adding the (b) activated ester solution with different volumes into the (a) DNA/RNA solution according to a set molar ratio, and carrying out vortex oscillation for 5 seconds to form solutions with different molar concentration ratios;
d. reacting for 10 minutes at 45 ℃ under 40KHZ ultrasonic frequency;
e. and (3) carrying out alcohol precipitation post-treatment, and comparing the molar yield of DNA/RNA after HPLC purification is carried out by adopting 0.1M triethylamine acetate and acetonitrile.
TABLE 11
TABLE 12
And (4) conclusion: in combination with the utilization efficiency of the isothiocyanate activated ester modified dye, the optimal molar ratio of the reaction of the DNA/RNA having an amino structure with the isothiocyanate activated ester modified dye is 1: 2.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (12)
1. A preparation method of a nucleic acid probe by an ultrasonic method is characterized in that nucleic acid with an amino active group and an activated ester modification dye capable of reacting with the amino group are subjected to an ultrasonic reaction in an alkaline buffer solution at the temperature of 30-50 ℃ to prepare the nucleic acid probe, wherein the frequency of the ultrasonic reaction is 35-50 KHZ;
the activated ester modified dye is a succinimide activated ester or isothiocyanate activated ester modified dye;
the ultrasonic reaction time is 5-15 minutes;
the ratio of the nucleic acid with the amino active group to the activated ester modified dye capable of reacting with the amino group is 1:1-1: 4; the activated ester modified dye is dissolved with an organic solvent.
2. The method according to claim 1, wherein the nucleic acid probe is prepared by ultrasonic reaction of the nucleic acid having an amino active group and the activated ester-modified dye capable of reacting with an amino group in an alkaline buffer solution at 45 ℃.
3. The method according to claim 1, wherein the nucleic acid is DNA or/and RNA.
4. The method of claim 1, wherein the ultrasonic reaction is carried out for a period of 5 to 10 minutes.
5. The method of claim 1, wherein the ultrasonic reaction has a frequency of 40 KHZ.
6. The method according to claim 1, wherein the ratio of the nucleic acid having an amino reactive group to the activated ester-modified dye reactive with an amino group is 1: 2.
7. The method according to claim 1, wherein the alkaline buffer solution has a pH of 7.1 to 10.0.
8. The method according to claim 7, wherein the alkaline buffer solution has a pH of8.5 of Na 2 CO 3 /NaHCO 3 And (4) a buffer solution.
9. The production method according to any one of claims 1 to 8, wherein after the ultrasonic reaction, an alcohol is added to the buffer solution for precipitation, followed by centrifugation, and the supernatant is removed to take the precipitate; washing the precipitate with alcohol to obtain the modified nucleic acid probe crude product.
10. The method according to claim 9, wherein the crude nucleic acid probe is dissolved in ultrapure water, and the solution is loaded on a high performance liquid chromatograph to purify the solution, thereby obtaining a pure nucleic acid probe.
11. The method according to claim 10, wherein the number of bases in the nucleic acid is not less than 22.
12. The method according to claim 11, wherein the number of bases in the nucleic acid is not less than 46.
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Address after: Building 23, No. 99, Shuhai Road, Songjiang District, Shanghai, 201611 Patentee after: Briggs Biotechnology (Shanghai) Co.,Ltd. Address before: Building 23, No. 99, Shuhai Road, Songjiang District, Shanghai, 201611 Patentee before: SHANGHAI BIOLIGO BIOTECHNOLOGY CO.,LTD. |