WO2021179394A1

WO2021179394A1 - Nucleic acid dye, preparation method therefor and use thereof

Info

Publication number: WO2021179394A1
Application number: PCT/CN2020/084324
Authority: WO
Inventors: 周杰; 郑慧敏; 马晓峰; 谈雪良; 钱近春; 夏继波
Original assignee: 苏州优逸兰迪生物科技有限公司
Priority date: 2020-03-13
Filing date: 2020-04-10
Publication date: 2021-09-16
Also published as: CN111363377B; CN111363377A

Abstract

A nucleic acid dye, a preparation method therefor and use thereof. The nucleic acid dye is a nucleic acid dye applicable to both agarose gel electrophoresis and polyacrylamide gel electrophoresis, and in particular, can be used for blue light imaging.

Description

Nucleic acid dye and its preparation method and application

Technical field

The invention is used for the common gel imaging dyeing of nucleic acid electrophoresis, and is specifically a novel nucleic acid dye that can be applied to agarose gel electrophoresis and polyacrylamide gel electrophoresis at the same time. It can especially be used for blue light imaging, and relates to the preparation and application of the dye. .

Background technique

Nucleic acid electrophoresis is an important method for nucleic acid research and an indispensable part of nucleic acid probes, nucleic acid amplification and sequence analysis. Nucleic acid electrophoresis usually uses two media: agarose gel and polyacrylamide gel. Agarose is a chain polysaccharide prepared from agar separation. Many agaroses coil together to form rope agarose bundles based on the action of hydrogen bonds and other forces, forming a large mesh gel, suitable for separation and purification of 200bp-50kB in length. The nucleic acid fragments can distinguish DNA fragments with a difference of 100bp; polyacrylamide gel is a very dense polymer with molecular sieve effect, concentration effect, and charge effect. It has the best separation effect for small fragments of DNA (5bp-500bp) good.

Commonly used nucleic acid electrophoresis dyes are EB, SYBR Green, SYBR Gold, GelRed and GelGreen, etc. The excitation wavelengths of different nucleic acid dyes are different. At present, gel imagers commonly used for nucleic acid electrophoresis are divided into two types of wavebands, namely, ultraviolet gel imaging systems and blue gel imagers (or blue gel cutters). Long-term ultraviolet irradiation is harmful to nucleic acid (DNA) and experimental personnel. Therefore, the future nucleic acid dyes tend to be safe and non-toxic blue nucleic acid dyes.

Existing nucleic acid dyes, such as the existing gelgreen dye, are safe, non-toxic, and highly sensitive, but they cannot be applied to polyacrylamide gel electrophoresis. The reason is: polyacrylamide gel density is a denser three-dimensional high polymer. This high-density polymer structure makes it difficult for fluorescent dyes as nucleic acid gel dyes to penetrate when separating nucleic acid molecules of different size ranges and different concentration ranges, resulting in poor nucleic acid staining. In addition, the existing dyes also have many limitations in their applications. For example, they are prone to tailing when dyeing and running electrophoresis before use, that is, they have a relatively large impact on the DNA migration rate and cannot guarantee the true level of DNA migration, which makes the experimental results appear. deviation. This kind of influence on the mobility, the displacement of the same strip will show obvious deviation.

Summary of the invention

technical problem

The present invention discloses a nucleic acid dye and its preparation method and application; as a nucleic acid dye, the compound of the present invention not only maintains the unique high sensitivity, but also is safer and non-toxic, especially solving the problem of existing nucleic acid dyes. The tailing phenomenon in the electrophoresis test can be applied to both agarose gel electrophoresis and polyacrylamide gel electrophoresis, especially with blue light imaging. The gel electrophoresis test results obtained under the same conditions of the compound of the present invention show that as a nucleic acid dye, the effect on the mobility of nucleic acid is minimal, and the accuracy and sensitivity of the electrophoresis test can be ensured.

The solution to the problem

Technical solutions

The present invention adopts the following technical solutions:

The chemical structure of nucleic acid dye is as follows:

Wherein, a, b, c, y, z are independently selected from 0-15; R _{1 is} selected from hydrogen, alkyl, cycloalkyl or (CH ₂ ) _t SO ₃ H, t is 1 to 5; R ^{1 is} selected Since or

, X is 1 to 5; preferably, a and b are independently selected from 0 to 14, c, y, and z are independently selected from 0 to 8; more preferably, a is selected from 1 to 12, and b is selected from 0 to 12. c is selected from 0 to 5, y is selected from 1 to 3, and z is selected from 1 to 3;

t and x are independently selected from an integer of 3 to 5;

R ₁ is a substituent, selected from hydrogen, alkyl or cycloalkyl; preferably, in the alkyl or cycloalkyl, the number of carbon atoms is less than 12;

B is a connecting bridge, selected from -O[(CH ₂ ) _n ]O-, -(CH ₂ ) _a1 -[O-(CH ₂ ) _y1 ] _b1 -[O-(CH ₂ ) _z1 ] _c1 -,- One of (CH ₂ ) _h -N-(CH ₂ ) _k -, -[(CH ₂ ) _n ]O[(CH ₂ ) _m ]-; where n is 1-12 and a1 is 1-12; y1 and z1 are independently selected from 1 to 5, preferably 2 to 3, b1 is 0 to 12, c1 is 0 to 5, h and k are independently selected from 1 to 12, and m is 1 to 12; or B contains 1 ~24 carbons and optionally contains at least one polymethylene unit selected from N, O heteroatoms or saturated 5-membered or 6-membered rings.

Y ^- is an anion, including but not limited to a halogen ion or sulfonate ion (OTs ^-).

Ultraviolet gel imaging system is currently the most popular nucleic acid gel imaging equipment on the market, but long-term ultraviolet irradiation is harmful to nucleic acids and experimenters. Therefore, the future development trend is to use safe blue gel imaging System or Blu-ray glue cutter. The dye of the present invention is a nucleic acid dye used for blue light imaging, which is suitable for blue gel imaging systems or blue light gel cutters and other equipment to eliminate the harm of imaging equipment to nucleic acids and experimenters.

Existing nucleic acid dyes such as SYBR Green, GelGreen and so on. Among them, SYBR Green, due to its small molecular weight, can enter living cells and bind to the DNA of human cells, causing nucleic acid mutations, and potentially carcinogenic risks to users. This potential hazard may gradually reduce the application market; and GelGreen The molecular weight is large, and the phenomenon of dispersion and tailing is easy to appear in agarose electrophoresis. As polyacrylamide gel is a denser three-dimensional polymer, it is difficult for the dye to penetrate and the nucleic acid staining effect is not good. Dyeing polyacrylamide nucleic acid gel with silver staining method, due to the large amount of silver nitrate and formaldehyde, not only caused serious pollution to the environment, but also seriously endangered human health. The invention designs a new dye structure, which eliminates the hidden danger of carcinogenesis, and also solves the phenomenon that the target bands appearing in the original dye are easily dispersed and the tail cannot be separated. At the same time, the dye can easily penetrate into the polyacrylamide gel. A high-density polymer.

The present invention discloses the application of the above-mentioned nucleic acid dye in nucleic acid blue gel imaging; or the application of the above-mentioned nucleic acid dye as nucleic acid blue gel imaging dye; or the application of the above-mentioned nucleic acid dye in preparing blue gel imaging reagent.

The present invention discloses a preparation method of the above-mentioned nucleic acid dye, which includes the following steps:

(1) Using 2-(methylthio)benzothiazole as a raw material, after a substitution reaction, intermediate I is obtained;

(2) Using 4-methylquinoline and bromo-polyethylene glycol-carboxylic acid as raw materials to prepare Intermediate II;

(3) Using Intermediate I and Intermediate II as raw materials to prepare Intermediate III;

(4) Reacting Intermediate III with a diamine compound to prepare a nucleic acid dye;

The chemical structure of Intermediate I is as follows:

The chemical structure of bromo-polyethylene glycol-carboxylic acid is as follows:

Br-(CH ₂ ) _a -[O-(CH ₂ )y] _b -[O-(CH ₂ )z] _c -COOH

The chemical structure of Intermediate II is as follows:

The chemical structure of Intermediate III is as follows:

The chemical structure of the diamine compound is as follows:

H ₂ NB-NH ₂ .

The substituents in the preparation method are the same as those in the chemical structural formula of nucleic acid dyes.

The present invention discloses a nucleic acid gel imaging method, which includes the following steps. The nucleic acid dye is mixed with an agarose solution to prepare an agarose gel, then placed in an electrophoresis tank for loading, then electrophoresis, and finally imaging. Complete nucleic acid gel imaging.

The invention discloses a nucleic acid gel imaging method, which includes the following steps: loading a sample into a polyacrylamide gel, then performing electrophoresis, and then immersing the electrophoresis gel in a bubble staining solution, and finally imaging to complete the nucleic acid gel Imaging; the bubble dye solution contains the above-mentioned nucleic acid dye.

In the nucleic acid gel imaging method disclosed in the present invention, agarose gel or polyacrylamide gel is the prior art, and the specific gel imaging process is also the prior art. The inventiveness of the present invention is that the above nucleic acid dye is disclosed, which is suitable for The agarose gel system is suitable for the polyacrylamide gel system, especially to solve the phenomenon that the existing dyes are prone to produce the purpose of the band dispersion and tailing can not be separated, the nucleic acid migration rate is affected, and it is safe for the human body and the environment. Non-toxic.

The beneficial effects of the invention

Beneficial effect

Compared with ultraviolet, blue light is the development trend of nucleic acid imaging, and the present invention is suitable for a blue gel imaging system or a blue gel cutter. Compared with common nucleic acid dyes in the market, the present invention can be used not only for agarose gel staining, but also for polyacrylamide nucleic acid gel staining, and solves the problem that some dyes are prone to produce target bands that are diffuse, tails and cannot be separated, and nucleic acid migration rate The phenomenon is affected, and at the same time, the present invention is safe and non-toxic to the human body and the environment.

Brief description of the drawings

Description of the drawings

Figure 1 is the mass spectrum of the compound No. 1 in Table 1 of the present invention;

Figure 2 is the mass spectrum of the compound No. 2 in Table 1 of the present invention;

Figure 3 is the mass spectrum of the compound No. 3 in Table 1 of the present invention;

Figure 4 is the mass spectrum of the compound No. 5 in Table 1 of the present invention;

Figure 5 is the mass spectrum of the compound No. 6 in Table 1 of the present invention;

Fig. 6 is an agarose gel diagram of the compound No. 1 in Table 1 of the present invention and the existing GelGreen dye;

Figure 7 is an agarose gel diagram of the compound numbered 12 in Table 1 of the present invention;

Figure 8 is a graph of polyacrylamide gel of existing UE Page GelRed dye;

Figure 9 is a diagram of the polyacrylamide gel of the compound numbered 1 in Table 1 of the present invention;

Figure 10 is a diagram of the polyacrylamide gel of compound No. 12 in Table 1 of the present invention;

Figure 11 is a graph of polyacrylamide gel of compound No. 5 in Table 1 of the present invention.

Invention embodiment

Embodiments of the present invention

Example 1 Preparation of nucleic acid dye

1. Add 25g 2-(methylthio)benzothiazole and 30.8g methyl p-toluenesulfonate to a 500mL reaction flask, stir mechanically, heat to 60°C, and track by TLC until the reaction is complete (developer: acetonitrile: water = 5:1); After the reaction, 300mL of ethyl acetate was added, the temperature was raised to 75°C and refluxed for 3 hours; then the temperature was reduced to room temperature, filtered, the filter cake was washed with ethyl acetate again, filtered, and the filter cake was vacuum dried to obtain 47g of intermediate product I.

in:

2-(Methylthio)benzothiazole:

Methyl p-toluenesulfonate:

Intermediate I:

2. Add 11.76g 4-methylquinoline, 28g bromo-triethylene glycol-carboxylic acid, 10mL o-dichlorobenzene into a 250mL reaction flask, heat to 120℃, and follow TLC to the end of the reaction (developing agent is : Acetonitrile: water = 5: 2); after the reaction, it was cooled to room temperature, ethyl acetate was added, washed twice, filtered, and dried in vacuum to obtain 30 g of Intermediate II.

in:

4-Methylquinoline:

Bromo-triethylene glycol-carboxylic acid:

Intermediate II:

3. Add 27g of Intermediate I, 30g of Intermediate II, 50mL of dimethylformamide DMF, 22mL of triethylamine TEA to a 500mL reaction flask, and follow TLC to the end of the reaction (developer: dichloromethane: methanol: glacial acetic acid = 5:1:0.1); after the reaction, the DMF was drained by a vacuum pump, dissolved in acetonitrile, and then added dropwise to ethyl acetate to precipitate a solid, filtered, and dried under vacuum for 48 hours to obtain 31 g of Intermediate III.

in:

Intermediate III:

4. Add 200mg Intermediate III, 3mL DMF, 150uL TEA to a 25mL reaction flask, stir for 15 minutes in an ice water bath, and add 130mg 2-succinimidyl-1,1,3,3-tetramethylurea tetrafluoroboron Ester TsTu (added in batches according to 40mg+30mg+20mg+20mg+20mg, 5 minutes interval for each batch), TLC tracked until the reaction of Intermediate III was complete (developer: dichloromethane: methanol: glacial acetic acid = 5:1 ：0.1); Dilute 10uL of ethylenediamine with DMF to 100uL and add it to the reaction flask (add it in batches according to 50uL+30uL+20uL), and add a drop of triethylamine, TLC track until the end of the reaction (developer: Dichloromethane: methanol: glacial acetic acid = 5:1: 0.1); after the reaction, drain DMF, wash with ethyl acetate once, purify alumina through column, eluent is acetonitrile: water = 98: 2, collect spin Dried and lyophilized to obtain 46 mg of the final product. See compound No. 1 in Table 1.

In this embodiment, the substitution reaction of step (1) is carried out in the presence of a sulfonic acid compound, such as methyl p-toluenesulfonate, and the obtained intermediate or product is an internal salt or a complex with a sulfonate ion as an anion. Further, the product with the sulfonate ion as the anion is replaced by the halogen ion to obtain the product with the halogen ion as the anion, the nucleic acid dye. Halogen ion replacement can be carried out in a halogen salt solution, which is a conventional technique.

Example 2 By replacing the raw materials, other nucleic acid dyes in Table 1 can be prepared, specifically conventional replacements.

For example, replacing the ethylenediamine in step (4) of Example 1 with the compound of formula ① or the compound of formula ② and the compound of formula ③, and the rest remain unchanged, to obtain the compounds of No. 2, No. 3, and No. 4 in Table 1, where The chemical formulas of the compound of formula ①, the compound of formula ②, and the compound of formula ③ are as follows: H ₂ N(CH ₂ CH ₂ O) ₃ CH ₂ CH ₂ NH ₂ , H ₂ NCH ₂ CH ₂ OCH ₂ CH ₂ NH ₂ , H ₂ NOCH ₂ CH ₂ ONH ₂ .

For example, the ethylenediamine in step (4) of Example 1 is replaced with the compound of formula 1 or the compound of formula 2, and the rest remains unchanged, to obtain the compound of No. 7 and No. 8 in Table 1, wherein the compound of formula 1 and the compound of formula 2 The compound structural formulas are as follows:

For example, the bromo-triethylene glycol-carboxylic acid in step (2) of Example 1 is replaced with the compound of formula ④, and the rest remains unchanged, to obtain the compound No. 5 in Table 1, wherein the compound of formula ④ is as follows:

The final product is subjected to anion replacement according to conventional methods to obtain dyes with different anion coordination. For example, the product of Example 1 is conventionally replaced in a saturated sodium chloride aqueous solution or sodium iodide aqueous solution, and is concentrated and filtered after conventional stirring. The solid is a dye whose anion is chloride or iodide:

The other compounds in Table 1 can be prepared on the basis of the above and according to conventional techniques.

The methyl p-toluenesulfonate in step (1) of the embodiment was replaced with 20.2 g of 1,3-propane sultone, and the rest remained unchanged to obtain the compound number 12.

Table 1 Fluorescent nucleic acid dyes

The mass spectra of compounds No. 1, No. 2, No. 3, No. 5, and No. 6 in Table 1 are shown in Fig. 1 to Fig. 5 respectively.

Weigh the solid dye of the present invention into water and stir to dissolve the dye completely to obtain a dye solution, which is used for the following gel imaging. In the nucleic acid gel imaging method disclosed in the present invention, agarose gel or polyacrylamide gel is the prior art, and the specific gel imaging process is also the prior art. The inventiveness of the present invention is that the above-mentioned nucleic acid dye is disclosed.

Example 3 Application of the dye of the present invention

(1) The configuration of the dye of the present invention

Weigh 5mg of the above-mentioned nucleic acid dye of the present invention, add 100μL of diH ₂ O to make it completely dissolve and determine the OD value, set as 10000x, directly _{dilute with diH 2} O to 1x as the working concentration, for example, add 2.5μL to 25mL gel. It is 1x working concentration; existing dyes are also formulated.

(2) The specific experimental method for the application of the product of the present invention in agarose gel electrophoresis to solve the phenomenon of smearing and tailing is as follows:

1. Weigh 0.6g of agarose powder into an Erlenmeyer flask;

2. Add 60mL of 1xTAE to the Erlenmeyer flask;

3. Heat it in a microwave oven and boil it repeatedly for three times to fully dissolve it. The final solution volume is 60mL;

4. Measure 25mL of agarose solution in a measuring bucket, and pour them into two Erlenmeyer flasks. One of the Erlenmeyer flasks is filled with 2.5μL of the control Biotium GelGreen (41005) dye solution, and one Erlenmeyer flask is filled with 2.5μL of the agarose solution. For the dye solution of the invention, shake the conical flask to mix the dye and agarose solution, and pour them into the prepared gel device respectively;

5. Gel at room temperature for 1 hour, (you can also place it at room temperature for 20 minutes until the gel does not shake, then put it in the refrigerator at 4°C and place it for 15 minutes to solidify);

6. Put the gelled agarose gel into the electrophoresis tank filled with the electrophoresis solution (1xTAE), and routinely load 5μL nucleic acid markers in the control gel (Gel Green) and the experimental group gel (the dye of the invention) in sequence (Existing conventional substances);

7. After the sample is loaded, turn on the electrophoresis tank switch, set the voltage to 160V, and the time is 30min, and start to run the electrophoresis;

8. After the electrophoresis is completed, take out the agarose, place it on the blue gel imaging system and the blue gel slicer, take a photo, and save the picture to the corresponding folder. The result is shown in Figure 6. Each figure is loaded in order from left to right. : 1. UE 100bp; 2. UE 2000 bp; 3. UE 5000 bp; 4. UE 1 kb; 5. UE 15000 bp; 6. YS 1 kb; 7. Takara 1 kb; 8. Vazyme 1 kb.

result:

1. Compared with the control group (Figure 6 left, Gelgreen) of the present invention (Figure 6 right, compound numbered 1 in Table 1), the background color is lighter and the bands are clear;

2. Compared with the control group, the large segment bands of the present invention can be clearly separated without the phenomenon of smearing tailing;

3. Explain that the present invention can effectively solve the phenomenon of dispersion and tailing in existing products.

Replace the compound numbered 1 in Table 1 with the compound numbered 12 in Table 1, and use the same gel imaging method to obtain the gel chromatogram, as shown in Figure 7, where the lanes are from left to right: 1.UE 2000bp 5ul; 2 .UE 15000bp 5ul; 3.DS 2000bp 10ul; 4.DS 15000bp 10ul; 5. Takara 2000bp 10ul; 6. Takara 15000bp 5ul.

(3) The specific experimental methods for the application of the product of the present invention in polyacrylamide gel electrophoresis are as follows:

1. After the glue plate of the gel device is assembled, add water and let it stand for 20 minutes to check the tightness of the device. If the tightness is better, pour clean water and prepare 2 pieces of 5% non-denaturing PAGE glue;

2. In a 50mL test tube, add H ₂ O 9.4mL, 30% Acrylamid 2.5mL, 5xTBE 3.0mL, and finally add 10% AP 0.11mL and TEMED 0.010mL and mix well (H ₂ O, 30% Acrylamid, 5xTBE in advance) Configured, 10% AP and TEMED need to be added before glue filling);

3. Add the mixed non-denatured glue, make the rubber sheet completely filled with liquid, and insert the comb (note the front and back sides, the smooth side of the comb is close to the thick rubber sheet side);

4. Solidify for 1 hour at room temperature to make the glue solidify completely;

5. Load 5μL of sample;

6. The electrophoresis buffer is 1xTBE, the voltage is set to 100V, the electrophoresis time is 60min, and the electrophoresis is started;

7. Configure the dye solution: Take 2 containers, add 100mL 0.1M NaCl solution (10mL 1M NaCl mixed with 90mL deionized water), respectively add 30μL of the control dye UE Page GelRed (S2005) and the present invention to the above 100mL solution The dyes are mixed evenly;

8. After the electrophoresis is completed, carefully remove the gel from the glass plate and put it in the bubble staining for 30 minutes;

9. After dyeing, put it on the blue gel imaging system, blue gel cutter, and ultraviolet gel imaging system, take a photo, and save the picture to the corresponding folder.

Control UE Page GelRed dye stained PAGE gel can only be viewed under ultraviolet light, but cannot be imaged under blue light, as shown in Figure 8. The lanes are from left to right: 1.UE 100bp; 2.UE 2000pb; 3.Takara 100bp; 4.Takara 500bp; 5. Takara 2000bp; 6. RNA.

The dye-stained PAGE gel of the present invention (the compound numbered as 1 in 1 in the table) can be imaged under blue light, as shown in Figure 9, the lanes from left to right: 1.UE 1kb 200ng; 2.UE 1kb 100ng; 3.UE 1kb 50ng ; 4. UE 1kb 25ng; 5. UE 100bp; 6. UE 2000bp; 7. UE 5000bp; 8. UE 1kb; 9. UE 15000bp.

result:

Compared with the control group, the present invention can stain the PAGE gel, and will not cause problems such as the mutation of nucleic acid induced by long-time UV irradiation. The dye will not cause any influence on the target band under blue light, and the band is clear .

The dye of the present invention is replaced with the existing gelgreen dye, and after the same polyacrylamide gel electrophoresis test as described above, no matter the blue light or the ultraviolet light, the band cannot be observed, and the imaging effect is very poor and cannot be observed.

Replace the compound numbered 1 in Table 1 with the compound numbered 12 in Table 1, and use the same gel imaging method to obtain PAGE gel chromatography, as shown in Figure 10. The lanes are from left to right: 1.UE 100bp 5ul; 2 .UE 2000bp 5ul; 3.DS 50bp 5ul; 4. Takara 100bp 5ul; 5. Takara 2000bp 5ul.

Replace the compound numbered 1 in Table 1 with the compound numbered 5 in Table 1, and use the same gel imaging method to obtain PAGE gel chromatography, as shown in Figure 11. The lanes are from left to right: 1. Takara 2000bp 1ul; 2 .Takara 2000bp 5ul; 3.Takara 2000bp 10ul; 4.Takara 2000bp 1ul; 5.Takara 2000bp 5ul; 6.Takara 2000bp 10ul; 7.UE 100bp 5ul; 8.UE 2000bp 5ul; 9.UE 5000bp 5ul; 10.UE 1kb 5ul; 11.UE 15000bp 5ul; 12. Genstar 1kb 5ul; 13. Takara 1kb 5ul; 14. Vzayme 1kb 5ul.

In summary, the present invention has developed a new type of nucleic acid dye, which integrates good dyeing effect, high safety, very low nucleic acid migration influence rate, wide applicability, and can be used for blue light imaging. It effectively solves the defect that existing nucleic acid dyes, such as gelgreen dyes, cannot be used in polyacrylamide gel electrophoresis due to poor dyeing effects, and especially solves the problem of tailing when the existing dyes are dyed and run electrophoresis before use. The migration rate of DNA has a relatively large impact, and the true level of DNA migration cannot be guaranteed, which results in deviations in the experimental results. For this impact on the mobility, the displacement of the same band will be significantly deviated.

Claims

The chemical structure of nucleic acid dye is as follows:

Wherein, a, b, c, y, z are independently selected from 0-15; R 1 is selected from hydrogen, alkyl, cycloalkyl or (CH 2 ) t SO 3 H, t is 1 to 5; R 1 is selected from (CH 2) x SO 3 - , x is 1 to 5.
The nucleic acid dye according to claim 1, wherein a and b are independently selected from 0-14, and c, y, and z are independently selected from 0-8.
The nucleic acid dye according to claim 2, wherein a is selected from 1-12, b is selected from 0-12, c is selected from 0-5, y is selected from 1-3, and z is selected from 1-3.
The nucleic acid dye according to claim 1, wherein B is a connecting bridge selected from -O[(CH 2 ) n ]O-, -(CH 2 ) a1 -[O-(CH 2 ) y1 ] b1- One of [O-(CH 2 ) z1 ] c1 -, -(CH 2 ) h -N-(CH 2 ) k -, -[(CH 2 ) n ]O[(CH 2 ) m ]-; Or B is a polymethylene unit containing 1-24 carbons and optionally containing at least one heteroatom selected from N and O or containing a saturated 5-membered ring or a 6-membered ring.
The nucleic acid dye according to claim 4, wherein n is 1-12, a1 is 1-12; y1 and z1 are independently selected from 1-5, b1 is 0-12, c1 is 0-5, h, k is independently selected from 1-12, and m is 1-12.
The use of the nucleic acid dye of claim 1 in nucleic acid blue gel imaging; or the use of the nucleic acid dye of claim 1 as a nucleic acid blue gel imaging dye; or the nucleic acid dye of claim 1 in the preparation of blue gel imaging reagents In the application.
The preparation method of nucleic acid dye according to claim 1, characterized in that it comprises the following steps:

(1) Using 2-(methylthio)benzothiazole as a raw material, after a substitution reaction, intermediate I is obtained;

(2) Using 4-methylquinoline and bromo-polyethylene glycol-carboxylic acid as raw materials to prepare Intermediate II;

(3) Using Intermediate I and Intermediate II as raw materials to prepare Intermediate III;

(4) Reacting Intermediate III with a diamine compound to prepare a nucleic acid dye;

The chemical structure of Intermediate I is as follows:

The chemical structure of Intermediate II is as follows:

The chemical structure of Intermediate III is as follows:

The chemical structure of the diamine compound is as follows:

H 2 NB-NH 2 .
The method for preparing nucleic acid dye according to claim 7, wherein a and b are independently selected from 0-14, and c, y, and z are independently selected from 0-8.
A method for nucleic acid gel imaging, comprising the steps of mixing the nucleic acid dye of claim 1 with an agarose solution to prepare an agarose gel, then placing it in an electrophoresis tank for loading, then performing electrophoresis, and finally imaging, Complete nucleic acid gel imaging;

Or a nucleic acid gel imaging method, including the following steps, loading the sample into a polyacrylamide gel, then performing electrophoresis, and then immersing the electrophoresed gel in a bubble staining solution, and finally imaging to complete the nucleic acid gel imaging; The foam dye solution contains the nucleic acid dye according to claim 1.
The method for imaging nucleic acid gel according to claim 9, wherein the imaging of the nucleic acid gel is performed by blue light irradiation.