CN113583467B - A nucleic acid fluorescent dye and its preparation and application - Google Patents

Abstract

The invention relates to the field of fluorescent dyes, in particular to a novel nucleic acid fluorescent dye. The invention provides a fluorescent dye, which has a structural formula shown as a formula I, wherein A^‑Is chloride ion, bromide ion or iodide ion. The invention carries out structural modification on the dye and provides another compound which has high sensitivity, good stability, easily obtained raw materials, simple synthesis, low sequence preference and small inhibition on PCR.

Description

A nucleic acid fluorescent dye and its preparation and application

technical field

The present invention relates to the field of fluorescent dyes, in particular to a novel nucleic acid fluorescent dye.

Background technique

Fluorescent dyes refer to fluorescent substances that can emit specific wavelengths after absorbing excitation light of a certain wavelength. Combining a fluorescent dye with a substance that does not emit fluorescence can selectively convert the chemical signal of the substance into a fluorescent signal that can be easily measured by analytical instruments, thereby realizing the fluorescence detection of a specific target. The detection method has the advantages of high sensitivity, good selectivity, visualization, and real-time, non-destructive online monitoring in vivo, so it has been widely used in the field of biological analysis.

Nucleic acids (such as DNA and RNA) are carriers of genetic information of living organisms, and are often used as target analytes due to their large amount of information and relatively stable content in cells. Currently, fluorescence detection is the mainstream method for nucleic acid analysis. In addition to labeling fluorescent dyes by covalent coupling, non-covalently bound "luminescent probes" (i.e. nucleic acid fluorescent dyes) are the most advantageous means for qualitative and quantitative analysis of nucleic acids. Fluorescent dyes can be non-covalently bound to nucleic acid molecules, and the detection of target nucleic acid molecules can be achieved through the huge difference in autofluorescence properties before and after binding. These fluorescent dyes are widely used for nucleic acid analysis in a variety of states, such as solution, gel electrophoresis, intracellular and environmental. At present, there are many fluorescent dyes for nucleic acid analysis on the market, among which the SYBR series dyes sold by Invitrogen Corporation of the United States have excellent performance and occupy most of the nucleic acid dyes market in the world.

Among the SYBR series of dyes, the most widely used is SYBR Green I, which has been widely used in routine DNA analysis, DNA gel staining and real-time quantitative PCR (qPCR) due to its low background, low toxicity and ultra-high sensitivity. Nucleic acid dyes for routine detection. However, SYBR Green I still has the following shortcomings:

(1) First, the dye applied to qPCR should have excellent chemical stability during PCR (105°C-55°C) and storage period (-20°C-4°C). This requires dyes and their complexes with nucleic acid molecules to have certain thermal stability. In addition, qPCR mostly uses Tris as a buffer, and the low temperature is alkaline and the high temperature is slightly acidic. Therefore, the dyes are also required to have good acid-base stability. However, SYBR Green I does not have good stability under the above conditions;

(2) SYBR Green I has a concentration-dependent inhibitory effect on the PCR process, and the use of high-concentration dyes can inhibit the PCR process;

(3) In routine analysis of nucleic acids (including qPCR and gel electrophoresis imaging), the sequence of dye-bound DNA should have low or no preference. But according to literature reports, SYBR Green I has obvious A-T sequence preference (see Zipper H, Brunner H, Bernhagen J, Vitzthum F (2004) Nucleic Acids Res 32:103e);

(4) according to the instructions for use provided by Invitrogen company, SYBR Green I can keep good stability in DMSO, and has poor stability in aqueous solution;

(5) In addition, although SYBR Green I has excellent fluorescence properties, its synthesis is extremely difficult and the yield is extremely low (less than 2%) according to retrosynthetic analysis. In addition, due to the high price of its raw materials, SYBR Green I is expensive (the specification is 10,000x, and the official price of 1 mL of the stock solution is as high as about 10,000 yuan), and the cost remains high in practical applications.

Although Invitrogen also sells another dye, SYBR Safe, which is less expensive and less mutagenic and can be used as an alternative to SYBR Green I, this alternative dye is not water-soluble and sensitive enough.

SUMMARY OF THE INVENTION

Aiming at the shortcomings of the high-efficiency nucleic acid fluorescent dyes (such as SYBR Green I) in the prior art, such as instability, high concentration affecting the PCR process, and difficulty in synthesis, the present invention carries out structural modification of the dyes to provide another kind of high sensitivity, good stability, and raw materials. Compounds that are readily available, simple to synthesize, have low sequence preference, and are less inhibitory to PCR.

Technical scheme of the present invention:

The first technical problem to be solved by the present invention is to provide a fluorescent dye, and the structural formula of the fluorescent dye is shown in formula I:

Wherein, A ^- is chloride ion, bromide ion or iodide ion.

The second technical problem to be solved by the present invention is to provide a preparation method of the above-mentioned fluorescent dye. The preparation method is as follows: the compound represented by the formula II and the monomer M are stirred and reacted in a solvent at room temperature for 6 to 24 hours to obtain a mixture, and then a mixture is obtained. The mixture is purified to obtain the fluorescent dye shown in formula I; wherein, the monomer M is propylamine substituted with an alkylamino group.

Further, in the above preparation method, the equivalent ratio of the compound represented by formula II to the monomer M is 1:1 to 1:5.

Further, in the above preparation method, the monomer M is selected from: N,N-dimethyl-1,3-diaminopropane or 3-diethylaminopropylamine.

Further, in the above preparation method, the solvent is ethanol or dichloromethane.

Further, in the above reaction, the stirring time is 6 to 12 hours.

Further, the compound shown in the formula II is prepared by the following method:

First prepare the compound represented by formula III

The compound represented by formula IV is then prepared

其中，所述溶剂1选自乙腈、二氯乙烷或二甲基甲酰胺中的至少一种；The compound represented by formula IV is reacted with phosphorus oxychloride in solvent 1 at 65°C to 85°C under reflux to obtain the compound represented by formula V

Wherein, the solvent 1 is selected from at least one of acetonitrile, ethylene dichloride or dimethylformamide;

The compound represented by formula V and the compound represented by formula III are mixed in solvent 2 with an equivalent ratio of 1:1 to 1:3 at room temperature to obtain the compound represented by formula II; the solvent 2 is ethanol or dichloromethane Methane.

Further, the compound shown in formula III is prepared by the following method: the 2-methylmercaptobenzothiazole (with or without a substituent) is subjected to a methylation reaction with a methylating reagent (such as methyl iodide) to obtain formula III compounds shown.

Further, the compound shown in formula IV is prepared by the following method: the compound shown in formula IV is prepared by 2-hydroxy-4-methylquinoline and iodobenzene or phenylboronic acid under the action of a catalyst; The catalysts are copper acetate, potassium carbonate and copper powder.

The third technical problem to be solved by the present invention is to provide the use of the above-mentioned fluorescent dyes, which can be used in qPCR analysis or gel electrophoresis imaging.

It is found for the first time in the present invention that when the compound represented by formula II and monomers such as N,N-dimethyl-1,3-diaminopropane are used as raw materials, a class of The novel fluorescent dye (Formula I), and the obtained fluorescent dye has higher nucleic acid molecule response sensitivity than SYBR Green I, has higher thermal stability after being combined with nucleic acid, and can also improve the melting temperature of nucleic acid molecules.

In addition, the benzothiazole and quinoline rings in the fluorescent dye of the present invention are connected by a methine bond, and the single bond therein can rotate freely, resulting in strong intramolecular torsional charge transfer (TICT) in the free state of the dye molecule. Moreover, due to the steric hindrance of the phenyl substituents and alkylamino substituents on the quinoline ring, the compactness of the aggregation and stacking of the dye molecules is reduced, and the dye monomer itself has almost no fluorescence. When the fluorescent dye is combined with a nucleic acid molecule, the methine bond connecting the benzothiazole and the quinoline ring is fixed by the restriction of the nucleic acid molecule structure, which facilitates the formation of a conjugated structure, thereby obtaining an enhanced fluorescent signal.

Beneficial effects of the present invention:

1) The present invention provides the design of a novel nucleic acid fluorescent dye, the raw materials for preparing such dyes are easily available, the purchase channels are numerous, the cost is low, and the synthesis conditions are simple and easy to synthesize (the reaction can be completed in a short time at room temperature), and No additional purification of the intermediate product is required.

2) The fluorescent dye obtained in the present invention has excellent fluorescence performance and good stability, and can be directly used in common PCR buffers (even pure water) without adding protective reagents; it can specifically identify the secondary structure of nucleic acid molecules ; The dye has good thermal stability after interacting with nucleic acid molecules, and can effectively increase the melting temperature of nucleic acid molecules.

3) The fluorescent dye obtained in the present invention has low sequence preference for nucleic acid molecules and high sensitivity. The highly sensitive detection of nucleic acid molecules in solution, gel, cell and environment can be achieved by detecting the fluorescent signal of the mixed system of dye and nucleic acid molecule.

Description of drawings:

FIG. 1 is the absorption spectrum and fluorescence spectrum of compound c of Example 1 of the present invention.

Figure 2 shows the relationship between the fluorescence intensity of the compound c (-■-), SYBR Green I (-●-) and SYBR Green I analogs (-▲-) of the present invention combined with different concentrations of double-stranded DNA and the corresponding double-stranded DNA concentration curve graph.

Figure 3A is a graph showing the relationship between the fluorescence intensity of the compound c (solid line) of the present invention and SYBR Green I (dotted line) combined with a double-stranded DNA sample dsDNA-2 against temperature, and Figure 3B is the corresponding melting curve of dsDNA-2.

Figure 4A is a graph showing the relationship between the fluorescence intensity of the compound c (solid line) of the present invention and SYBR Green I (dotted line) binding to double-stranded DNA sample Harpin-1 versus temperature, and Figure 4B is the corresponding melting curve of Harpin-1.

Figure 5A is a graph showing the relationship between the fluorescence intensity of compound c (solid line) of the present invention and SYBR Green I (dotted line) binding to double-stranded DNA sample dsDNA-3 versus temperature, and Figure 5B is the corresponding melting curve of dsDNA-3.

Figure 6 is a bar graph showing the change of the fluorescence response of compound c and SYBR Green I to double-stranded DNA with different d(A-T) contents.

The left picture of Fig. 7 is the qPCR fluorescence amplification curve of the compound c of the present invention at the concentration of 0.5, 1, 2, and 4 μmol/L, and the right picture of Fig. 7 is the SYBR Green I at 0.5(0.55x), 1(1.1x), 2 qPCR fluorescence amplification curves at (2.2x) and 4 (4.4x) μmol/L concentrations.

The upper picture of Fig. 8 is the qPCR amplification curve of 1 μmol/L compound c of the present invention at the primer concentrations of 50, 100, 200, and 300 nmol/L, and the lower picture of Fig. 8 is that 1 μmol/L (1.1x) SYBR Green I at the same qPCR amplification curve under conditions.

The upper picture of Fig. 9 is the melting curve of the amplification product of 1 μmol/L compound c of the present invention at primer concentrations of 50, 100, 200 and 300 nmol/L, and the lower picture of Fig. 8 is that 1 μmol/L (1.1x) SYBR Green I Melting curves of amplification products under the same conditions.

Figure 10 is a comparison chart of the gel imaging effect of standard molecular weight DNA under the pre-staining mode, the left picture is 1 μmol/L (1.1×) SYBR Green I and the right picture is 1 μmol/L of the compound c of the present invention.

Figure 11 is a comparison chart of the gel imaging effect of standard molecular weight DNA under post-staining mode, the left picture is 1 μmol/L (1.1×) SYBR Green I and the right picture is 1 μmol/L of compound c of the present invention.

The left picture of Fig. 12 is a comparison chart of the gel imaging effect of 1 μmol/L (1.1x) SYBR Green I and 1 μmol/L compound c of the present invention on PCR amplification products in the pre-staining mode, and the right picture of Fig. 12 is the post-staining mode. Comparison of gel imaging effects.

FIG. 13 is a graph showing the relationship between the fluorescence intensity detected at 55° C. of the compound c of the present invention and the number of thermal cycles experienced.

Figure 14A is a graph showing the relationship between the absorbance of the present compound c (-•-) and SYBR Green I (-•-) in pure water versus time, and Figure 14B is the present compound c (-•-) and SYBR Green I ( -■-) Graph of absorbance versus time in Tris-HCl buffer solution.

Fig. 15 The synthetic route and cost comparison diagram of compound c and SYBR Green I.

Detailed ways

The fluorescent dye of the present invention can be prepared by the following preparation method, and the preparation method comprises the following steps:

1) First use methylating reagent (such as methyl iodide) to carry out methylation reaction on 2-methylmercaptobenzothiazole (with or without substituent) to obtain compound a

2) 2-hydroxy-4-methylquinoline and phenylboronic acid are obtained under the catalysis of copper acetate to obtain compound b

与三氯氧磷混合，得到化合物

随后将其与化合物

混合得到化合物

其与N,N-二甲基-1,3-二氨基丙烷反应，可得到最终的染料结构

3) will

Mix with phosphorus oxychloride to give compound

Then combine it with the compound

compound to mix

It reacts with N,N-dimethyl-1,3-diaminopropane to give the final dye structure

The specific reaction formula is as follows:

The specific embodiments of the present invention will be further described below with reference to the examples, but the present invention is not limited to the scope of the described examples.

Example 1: Preparation of the nucleic acid fluorescent dye of the present invention

The preparation method is as follows:

1) The raw material 2-Methylmercaptobenzothiazole (1.5g, 8.3mmol, 1eq) was dissolved in 30mL of dry ethanol, iodomethane (1.4g, 10mmol, 1.2eq) was added, and the reaction was carried out at 60°C for 5 hours. After the reaction is completed, remove the solvent, use petroleum ether to wash repeatedly, utilize the method of suction filtration to collect off-white solid product a:

2) The raw materials 2-hydroxy-4-methylquinoline (3.0g, 18.8mmol, 1eq), phenylboronic acid (2.3g, 18.8mmol, 1eq) and copper acetate (3.75g, 18.8mmol, 1eq) were dissolved in 150 -250mL of dichloromethane, after stirring at room temperature for 48-96h, suction filtration under reduced pressure. The filtrate was extracted three times with water, and the dichloromethane phase extract was taken, and dried by adding anhydrous calcium chloride for 1-2 hours. _The extract was concentrated and purified by SiO column to give a white solid b:

3) Dissolve the product b (100 mg, 0.4 mmol, 1 eq) in 30 mL of acetonitrile, add phosphorus oxychloride (184 mg, 1.2 mmol, 3 eq), and reflux at 60-85 °C for 12-24 h under nitrogen atmosphere. After the reaction is completed, cool to room temperature, remove the solvent, dissolve with compound a in 20-30 mL of dichloromethane, and add triethylamine dropwise with stirring at room temperature until no obvious white smoke emerges. After stirring at room temperature for 5 h, the raw material N,N-dimethyl-1,3-diaminopropane (122 mg, 1.2 mmol, 3 eq) was added dropwise, stirring was continued at room temperature for 6-24 h, the reaction mixture was concentrated, and purified and separated through a SiO ₂ column. , and recrystallized in dichloromethane and petroleum ether solvent to obtain pure compound c.

Example 2: Absorption spectrum and fluorescence spectrum of compound c

The DNA samples were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and were two complementary oligonucleotide chains, as shown in Table 1. DNA samples were purified by HPLC, and appropriate amount of DNA was dissolved in TE buffer (pH 7.4, 10 mmol/L Tris, 1 mmol/L EDTA) according to the instructions to prepare a stock solution of corresponding concentration, and stored at 4°C. During use, the two single strands (Pro-20 and Tar-20) of the same concentration were added to form double-stranded DNA (dsDNA-1).

Table 1

The absorption spectrum of compound c (1 μmol/L) and the fluorescence emission spectrum after binding with dsDNA-1 (20 bp, 100 nmol/L) were detected in TE buffer (shown in FIG. 1 ). The maximum absorption peak of compound c is 467 nm (blue light), and the fluorescence maximum emission peak after binding to DNA is located at 505 nm (green light). Compound c can be excited by a light source in the range of 400nm-500nm, which matches the optical channel of the mainstream qPCR and chemiluminescence imagers on the market, which shows that the dye can be well adapted to the existing instruments.

Example 3: Compound c for routine nucleic acid molecule quantification

DNA samples were the same as in Example 2.

以及被甲基封闭后的SYBR Green I类似物

对不同 浓度核酸分子的荧光响应灵敏度。在体积为200μL，TE缓冲液中，以5nmol/L为浓度梯 度，分别配置含有0-75nmol/L终浓度的dsDNA-1与终浓度为0.5μmol/L的化合物c、SYBR Green I(0.55x)及SYBR Green I类似物的混合液。使用荧光光谱仪Fluorolog-3(HORIBA， 美国)检测了上述混合物的荧光，如图2所示，以探测到的荧光强度对dsDNA-1浓度作图。 首先可以看出，dsDNA-1浓度为0时，几乎没有检测到荧光信号响应。随着dsDNA浓度 的增加，在一定范围内荧光对DNA浓度变化呈线性；而DNA浓度较高时，响应变为非线 性，信号逐渐趋于饱和。线性变化范围内的上升区域斜率代表染料分子对DNA浓度变化 的响应灵敏度。结果显示，化合物c对5nmol/L的dsDNA-1的响应灵敏度明显高于SYBR Green I及SYBR Green I的类似物。其次，对比相同浓度的染料与dsDNA-1结合饱和后的 荧光强度，化合物c的饱和荧光强度要明显高于SYBR Green I和SYBR Green I类似物。 以上结果表明：相比于SYBRGreen I以及SYBR Green I类似物，化合物c的结构具有独 特的优势，对DNA分子具有更好的结合能力和更高的检测灵敏度。Comparing the structure of compound c and compound c after the NH bond of compound c is blocked by n-propyl group SYBR Green I

and SYBR Green I analogs blocked by methyl

Fluorescence response sensitivity to different concentrations of nucleic acid molecules. In TE buffer with a volume of 200 μL and a concentration gradient of 5 nmol/L, dsDNA-1 containing a final concentration of 0-75 nmol/L and compound c and SYBR Green I (0.55x ) and a mixture of SYBR Green I analogs. The fluorescence of the above mixture was detected using a fluorescence spectrometer Fluorolog-3 (HORIBA, USA), as shown in Figure 2, and the detected fluorescence intensity was plotted against dsDNA-1 concentration. First, it can be seen that at 0 dsDNA-1 concentration, almost no fluorescence signal response was detected. With the increase of the dsDNA concentration, the fluorescence changes linearly with the DNA concentration in a certain range; however, when the DNA concentration is higher, the response becomes nonlinear and the signal gradually tends to be saturated. The slope of the rising region within the linear variation range represents the sensitivity of the response of the dye molecule to changes in DNA concentration. The results showed that the response sensitivity of compound c to 5nmol/L dsDNA-1 was significantly higher than that of SYBR Green I and SYBR Green I analogs. Secondly, comparing the fluorescence intensity of the same concentration of dye after binding to dsDNA-1, the saturated fluorescence intensity of compound c was significantly higher than that of SYBR Green I and SYBR Green I analogs. The above results show that compared with SYBRGreen I and SYBR Green I analogs, the structure of compound c has unique advantages, and has better binding ability and higher detection sensitivity to DNA molecules.

Example 4: Thermal stability of compound c combined with nucleic acid molecules

The preparation of DNA samples is the same as that in Example 2, and the DNA sequence used is shown in Table 2.

Table 2

Thermal stability assay protocol: The samples containing 1 μmol/L of compound c and 100 nmol/L of DNA were kept at 95 °C for 15 s with a volume of 20 μL, pH=8.3 Tris-HCl buffer (containing Mg ²⁺ ) and then annealed; After incubating at 45°C for 15min, use the StepOnePlus ^™ qPCR instrument (ABI, USA) to collect fluorescence signals at every +0.5°C (including 45°C); raise the temperature to 95°C and hold for 30s (Thermal stability test conditions of SYBR Green I and compounds c remains the same). Plot the fluorescence intensity of compound c and SYBRGreen I and the above-mentioned different DNA samples to form complexes with the curve of temperature (Figure 3A, Figure 4A and Figure 5A), and draw the melting curve of different DNA samples after taking the negative reciprocal of the slope of each point ( 3B, 4B and 5B). The test results show that: on the one hand, after the compound c prepared in Example 1 of the present invention is combined with DNA, the rate of decrease of the fluorescence intensity during the heating process (a relatively gentle change region before the change of the fluorescence intensity changes sharply) is significantly slower than that of SYBR Green I, This indicated that the thermal stability of the complex formed by compound c and DNA was significantly improved compared with SYBR Green I. On the other hand, comparing the abscissas (that is, melting temperature value, _Tm value) corresponding to the peaks of the melting curves of different DNA samples, the _Tm value detected by compound c is also greater than that of SYBR Green I (as shown in Table 3), and further It was confirmed that the complex of compound c and DNA has good binding ability and thermal stability.

table 3

Example 5: Sequence selectivity of compound c

The preparation of DNA samples is the same as that in Example 2, and the DNA sequence used is shown in Table 4.

Table 4

The DNA sample (AT-1) containing 10% adenine (A)-thymine (T) and the DNA sample (AT-1) containing 100% adenine (A) were prepared in a volume of 1 mL, pH=7.4 TE buffer with a concentration of 100 nmol/L. )-thymine (T) DNA sample (AT-2), to which compound c was added at a final concentration of 1 μmol/L. After incubation for 10 min, the fluorescence intensity of the complex formed by compound c and two different DNA samples was measured using a fluorescence spectrometer Fluorolog-3 (HORIBA, USA). As shown in Figure 6. The signal response difference of compound c to A-T sequence preference is only 15%, which is significantly lower than that of SYBR Green I (40%), indicating that compound c has very low sequence preference to nucleic acid molecules and is suitable for routine detection of nucleic acid molecules.

Example 6: Application of compound c in qPCR amplification

Using Compound c prepared in Example 1 as a fluorescent dye, StepOnePlus ^™ qPCR instrument (ABI, USA) was used to amplify the invA gene in Salmonella. The primer sequences amplified by qPCR are as follows:

Upstream primer: 5'-TCCCTTTCCAGTACGCTTCG-3'

Downstream primer: 5'-TCTGGATGGTATGCCCGGTA-3'

Template gene acquisition: use a bacterial DNA extraction kit (TIANamp Bacteria DNA Kit, item number: DP302, TIANGEN, Beijing) to extract Salmonella DNA according to the instructions.

qPCR conditions: hold at 95°C for 20s, incubate at 55°C for 30s, set the test fluorescence intensity at 55°C, and then extend at 72°C for 30s, for a total of 30-40 cycles.

A PCR kit (TaKaRa Taq ^TM HS Low DNA, Cat. No.: R090A, TaKaRa, Japan) was used to prepare a reaction system with a volume of 20 μL according to the instruction manual, including Taq ^TM HS Low DNA (2x), 10 μL; upstream primer and downstream primer 0.4 μL each μL (with final concentrations of 50 nmol/L, 100 nmol/L, 200 nmol/L and 300 nmol/L, respectively, DNA); Compound c or SYBR Green I for comparison, 2 μL (with final concentrations of 0.5 μmol/L, 1 μmol/L, 2 μmol/L and 4 μmol/L); template gene, 2 μL; add sterile water to make up to a final volume of 20 μL.

1) Compare the signal intensity of compound c and SYBR Green I in qPCR

Keeping other conditions the same, the qPCR fluorescence amplification curve (fluorescence intensity versus cycle number) of only changing the concentration of compound c is shown in the left panel of Figure 7, and the different concentrations of SYBR Green I used for comparison are shown in the right panel of Figure 7. The results showed that the amplitude and intensity of the fluorescence signal changes monitored by different concentrations of compound c were higher than those of SYBR GreenI at the corresponding concentrations. In addition, at higher concentrations, SYBR Green I showed a more obvious concentration inhibition effect, and compared with compound c at the same concentration, the _CT value lag (about 1 cycle). The experiment confirmed that compared with SYBR Green I, compound c can be used for real-time amplification detection of nucleic acid in a larger concentration range, and can achieve more sensitive detection.

2) Compare the amplification curves of compound c and SYBR Green I in qPCR

Keep other conditions the same (the concentrations of compound c and SYBR Green I are both 1 μmol/L), and only change the qPCR amplification curve of the primer concentration (the ordinate ΔR _n versus the number of cycles on the abscissa) as shown in the upper figure of Figure 8, using Different concentrations of SYBR Green I for comparison are shown in the lower panel of Figure 8. The results show that the compound c synthesized by the present invention has a lower amplification background, and also has no obvious concentration dependence compared with SYBR Green I at a low primer concentration (50 nmol/L).

3) Compound c monitors the melting temperature of PCR amplicons

SYBR Green I has been reported to be a useful tool for monitoring the amplification product (ie, the melting temperature of the amplicon) after qPCR amplification is complete. Determining the melting temperature of the amplicon can provide valuable information such as whether there is non-specific amplification and whether the amplicon is single. Accordingly, the concentration of the primers was changed at the same time, and the melting curve of the amplicon was monitored with compound c in the present invention (the upper figure in FIG. 9 ), to test the effect of monitoring the melting peak of the amplicon with compound c, and with SYBR Green I ( Figure 9 bottom) for comparison. First of all, there is a good correlation between the two amplicon melting temperature test results, and the measurement results are consistent. Secondly, compared with SYBR Green I, the test baseline of compound c is lower, indicating that compound c provided in Example 1 of the present invention can detect the melting temperature of amplicon.

Example 7: Comparative experiment of gel dyeing effect

According to reports, gel staining is currently mainly achieved in two ways: (1) before separation, the dye is pre-added to the DNA mixture for electrophoretic separation and then fluorescent imaging is performed, that is, the pre-staining method; (2) the DNA mixture is electrophoresed After separation, the gel is immersed in an aqueous solution or buffer of fluorescent dye for a period of time for fluorescence imaging, that is, post-staining; both methods are very common. Compound c (final concentration: 1 μmol/L) and SYBR Green I (final concentration: 1 μmol/L, 1.1×) prepared in Example 1 were used to effect the pre-staining method (Fig. 10) and the post-staining method (Fig. 11) respectively. In contrast, the DNA sample used was a 25-500bp DNA molecular weight standard Marker (Cat. No. B600303, BBI, USA), and the loading amount was controlled to be 10ng, 5ng, 2ng, 1ng and 0.5ng according to the instruction manual.

It can be seen from the comparison of the effects of the pre-dyeing method (Fig. 10) and the post-dyeing method (Fig. 11) that the two dyeing methods of compound c prepared in Example 1 of the present invention are comparable to SYBR Green I, and in the pre-dyeing method, Compared with SYBR Green I, there is no obvious tailing phenomenon in the staining result of compound c, and the band is cleaner. In addition, compound c (final concentration of 1 μmol/L) and SYBR Green I (final concentration of 1 μmol/L, 1.1×) were also compared for pre-staining and post-staining of the amplicon samples generated in Example 6. Comparing the effects (Fig. 12), the results show that in the process of practical application, compound c has the same effect as SYBRGreen I regardless of pre-dyeing or post-dyeing. And more obviously, compared with SYBR Green I compound c pre-stained with no tailing phenomenon in the actual sample, the band is cleaner and clearer, and the distinction between different DNA fragments is more obvious.

Example 8: Intrinsic stability of compound c

Generally, the temperature range of qPCR is 55℃-105℃, and the qPCR reagents should be stored at -20℃-4℃. In addition, nucleic acid molecules need to be in a pure aqueous or buffered solution environment. This requires the dye molecule to be stable in water or buffer, and also stable in the temperature range of 55°C-105°C and -20°C-4°C.

1) Thermal stability

In a system with a volume of 20 μL, containing compound c with a final concentration of 10 μmol/L, and Tris-HCl buffer (containing Mg ²⁺ ) at pH=8.3, compound c was kept at 95°C for 20s, 55°C for 30s, 72°C After 40 thermal cycles of 30 s incubation at °C, as shown in Figure 13, it remained stable (the test fluorescence intensity was set at 55 °C), and the signal did not decrease significantly, confirming the durability of compound c in qPCR.

2) Stability in solution

In a system with a volume of 3 mL, a pH=8.3 Tris-HCl buffer (containing Mg ²⁺ ) or pure water containing compound c or SYBR Green I (1.1×) at a final concentration of 1 μmol/L, every 12- The signal changes of compound c and SYBR Green I at their respective maximum absorption wavelengths were monitored for 24 hours, as shown in Figure 14A in pure water and Figure 14B in buffer solution. The results show that: SYBRGreen I in pure water is extremely unstable compared with buffer, which is consistent with the reported phenomenon, nearly half of the molecules are decomposed after 72 hours; All buffers are more stable than SYBR Green I. More importantly, compound c is very stable in pure water, which indicates that the storage environment of compound c is not as harsh as that of SYBR Green I. When the post-staining method is used for gel staining, it can be directly prepared with pure water without additional buffer configuration. , it is more convenient to use.

Example 9: Comparison of synthetic cost of compound c and SYBR Green I

At present, SYBR Green I is the most widely used and relatively cost-effective dye on the market. However, according to the analysis of the synthetic route of SYBR Green I (Fig. 15), the reaction conditions of compound c are low, there are many purchasing channels for raw materials, there is no need for self-synthesis, and the price is cheap (reagent price query website: https://www.bidepharm.com; https://www.bidepharm.com; https://www.bidepharm.com; https://www.bidepharm.com ://www.chemicalbook.com/ProductIndex.aspx); only three steps are required, no intermediate product purification is required, no heating is required for the key step reaction, and the yield is higher, and the above experiments have shown that the performance of compound c is better than SYBR Green I.

More importantly, in the actual nucleic acid molecule detection, the cost of dye is the most important part that affects the total cost of the experiment. Although SYBR Green I is a highly sensitive nucleic acid dye, it is expensive. The official price is about 10,000 yuan per 1mL (10,000x). The cost of the reagent itself is too high, especially for large-scale nucleic acid detection and gel electrophoresis imaging. As in the gel staining experiment of Example 7, it has been confirmed that compound c not only has the same staining effect as SYBR Green I, but even better effect, but the synthesis cost of compound c is far lower than that of SYBR Green I. This shows that compound c can greatly reduce the matching production cost under the condition of maintaining high sensitivity.

SEQUENCE LISTING

<110> Sichuan University

<120> A nucleic acid fluorescent dye and its preparation and application

<130> 2021.8.2

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 1

acagacacct acagacacct 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

aggtgtctgt aggtgtctgt 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

ctctcagtct gcatctcact 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

agtgagatgc agactgagag 20

<210> 5

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 5

acagacacct acagacacct acagacacct acagacacct acagacacct acagacacct 60

<210> 6

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 6

aggtgtctgt aggtgtctgt aggtgtctgt aggtgtctgt aggtgtctgt aggtgtctgt 60

<210> 7

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 7

gcagacacct acagacacct aaaaaaatag gtgtctgtag gtgtctgc 48

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

tccgcgcgcc cgggccgcca 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 9

tggcggcccg ggcgcgcgga 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 10

aaaaaaaaaa aaaaaaaaaa 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 11

tttttttttt tttttttttt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 12

tccctttcca gtacgcttcg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 13

tctggatggt atgcccggta 20

Claims (10)

1. A fluorescent dye, wherein the structural formula of the fluorescent dye is shown as formula I:

wherein A is^-Is chloride ion, bromide ion or iodide ion.

2. The method of preparing a fluorescent dye according to claim 1, wherein the method comprises: stirring and reacting the compound shown in the formula II and the monomer M in a solvent at room temperature for 6-24 hours to obtain a mixture, and then purifying the mixture to obtain the fluorescent dye shown in the formula I; wherein the monomer M is propylamine substituted by alkylamino;

3. the method of claim 2, wherein the equivalent ratio of the compound of formula II to monomer M is 1: 1-1: 5.

4. the method of claim 2 or 3, wherein the monomer M is selected from the group consisting of: n, N-dimethyl-1, 3-diaminopropane or 3-diethylaminopropylamine.

5. The method of claim 2 or 3, wherein the solvent is ethanol or dichloromethane.

6. The method for preparing a fluorescent dye according to claim 2 or 3, wherein the stirring time is 6 to 12 hours.

7. The method for preparing a fluorescent dye according to claim 2 or 3, wherein the compound represented by the formula II is prepared by the following method:

firstly, preparing the compound shown as the formula III

Then preparing the compound shown as the formula IV

Carrying out reflux reaction on a compound shown as a formula IV and phosphorus oxychloride in a solvent 1 at 65-85 ℃ to obtain a compound shown as a formula V

Wherein the solvent 1 is selected from at least one of acetonitrile, dichloroethane or dimethylformamide;

in a solvent 2, a compound shown as a formula V and a compound shown as a formula III are mixed in an equivalent ratio of 1: 1-1: 3 mixing at room temperature to obtain a compound shown as a formula II; the solvent 2 is ethanol or dichloromethane.

8. The method of claim 7, wherein the compound of formula III is prepared by the following method: and (3) carrying out methylation reaction on the 2-methylmercaptobenzothiazole by using a methylation reagent to obtain the compound shown in the formula III.

9. The method of claim 7, wherein the compound of formula iv is prepared by the following method: preparing a compound shown in a formula IV by reacting 2-hydroxy-4-methylquinoline with iodobenzene or phenylboronic acid under the action of a catalyst; wherein the catalyst is copper acetate or copper powder.

10. Use of a fluorescent dye in qPCR analysis or gel electrophoresis imaging, the fluorescent dye being as defined in claim 1 or prepared by the method of any one of claims 2 to 9.

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