CN110426442B

CN110426442B - Method for testing nucleic acid gel electrophoresis

Info

Publication number: CN110426442B
Application number: CN201910306043.5A
Authority: CN
Inventors: 夏继波; 焦兆友; 聂承斌; 钱近春; 谈雪良; 赵育明
Original assignee: Suzhou Youyi Landi Biotechnology Co ltd
Current assignee: Suzhou Youyi landi Biotechnology Co.,Ltd.
Priority date: 2016-06-13
Filing date: 2016-06-13
Publication date: 2022-02-11
Anticipated expiration: 2036-06-13
Also published as: CN110426442A; CN106146398A; CN106146398B

Abstract

The invention discloses a method for testing nucleic acid gel electrophoresis, wherein a dye compound is adopted to have a bridged symmetrical structure, the molecular structure is stable, the carried charge is large, the molecular weight is large, the preparation method is simple and controllable, and when the dye is applied as a nucleic acid dye, the dye has high sensitivity and almost has no influence on the mobility of DNA, so that the true level of DNA migration can be ensured, and the true test result can be reflected; when the fluorescent dye is used for gel test, the problem of obvious fluorescence quenching caused by the fact that the existing fluorescent dye can form a dimer in a high-concentration aqueous solution is solved, and the compound provided by the invention has a large amount of carried charges and a large molecular weight, so that the compound cannot penetrate through a cell membrane to enter a human body to cause damage, namely is nontoxic. Therefore, the invention is a nucleic acid gel electrophoresis test method with high sensitivity, safety and no toxicity.

Description

Method for testing nucleic acid gel electrophoresis

The invention belongs to a divisional application of invention application with the application date of 2016, 6 and 13 and the application number of 2016104106305, belonging to the part of test methods.

Technical Field

The invention belongs to the technical field of biological materials, particularly relates to a compound and application thereof as a low-mobility nucleic acid dye, and more particularly relates to a method for testing nucleic acid gel electrophoresis.

Background

The agarose (nucleic acid) gel electrophoresis is widely applied to molecular biology, and provides powerful means for gene recombination, molecular hybridization, DNA sequence research and the like. Fluorescent dyes have found widespread use as nucleic acid probes in the last decade. These fluorescent nucleic acid dyes can be used not only for electrophoretic nucleic acid gel staining, but also for quantitative detection of DNA in real-time polymerase chain reaction (i.e., qPCR). In terms of chemical structure, these nucleic acid fluorescent dyes can be mainly classified into acridine, phenanthridine, cyanine, fluorescein, rhodamine, and the like. EB (Ethidium bromide), a highly sensitive nucleic acid fluorescent dye, was used to visualize DNA in agarose and polyacrylamide gels. In the agarose (nucleic acid) gel electrophoresis process, the most commonly used nucleic acid staining agent is Ethidium Bromide (EB), which has good staining effect and convenient operation, but has poor stability and very high toxicity (carcinogenicity), and EB is a strong mutagen and has high carcinogenicity. Based on the molecular structure of EB, based on fully utilizing the fluorescent group (namely phenanthridinium) of EB molecules, structural change is carried out on EB, but the nucleic acid dyeing effect better than that of EB is reserved and even achieved. These novel nucleic acid dyes also have the advantage of lower toxicity compared to EB. Nucleic acid stains are also known as "edible salts" in molecular biology laboratories. As nucleic acid dyes capable of replacing EB, there are mainly two products, a compound prepared by connecting two EB luminescent groups (phenyl-phenonthridine) with one small molecule by betwen betheyt wu gmbh (biotium, Inc.) and a SYBR Green series dye produced by Molecular Probes, Inc. SYBR series dyes have the defects of not strong enough sensitivity, low toxicity and the like. The product of Beautty Umu stock Co., USA is nontoxic and has high sensitivity, but has larger influence on the mobility of DNA, namely the true level of DNA migration can not be ensured, thereby causing the deviation of experimental results. This effect on mobility, the same band shift will be significantly biased.

Disclosure of Invention

The invention aims to disclose a compound and an application thereof as a low-mobility nucleic acid dye; under the same conditions, compared with the prior art, such as the product of Beautto Uygur Co., Ltd, the compound of the invention not only keeps the characteristics of no toxicity and high sensitivity, but also has low influence on the mobility of DNA as a low-mobility nucleic acid dye.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a compound comprising the following chemical structure:

wherein the chemical structural formula of B is as follows:

wherein a, b, c, d, e and f are independently selected from integers between 0 and 12.

In the compound of the invention, the bridge contains a proper number of non-hydrogen atoms, wherein a, b, c, d, e and f are independently selected from integers between 0 and 12, R is-SO₃H、-SO₃ ^-Or H.

The compounds of the present invention may also be in the form of a compound salt having the following chemical structure:

wherein R' is anion, generally anion such as chlorine, bromine, iodine, etc.; the bridge may be an amide bond linkage or a non-amide bond linkage, or a bridge comprising X,x is O, N, NHCH₃、NCH₃CH₃And a branched alkane having an S atom. The anion does not influence the property of the compound, especially does not influence the special high sensitivity of the compound as nucleic acid dye, and has safer and nontoxic effect.

When the compound is used as a nucleic acid dye, compared with the existing products, the compound not only maintains the special high sensitivity, but also is safer and nontoxic, and particularly solves the tailing phenomenon in the prior nucleic acid electrophoresis. The invention further discloses the application of the compound as a nucleic acid dye.

According to the description of the embodiment of the invention, compared with the existing product, the compound disclosed by the invention not only has equivalent or even higher dye brightness, but also can obtain higher dyeing effect; as can be seen from an electrophoresis chart, the electrophoresis of the existing product has obvious influence on the nucleic acid electrophoresis mobility with different sample amounts, and the gel electrophoresis test result obtained by the compound of the invention under the same condition shows that the influence of the compound as a nucleic acid dye on the nucleic acid mobility is very little, so that the accuracy and the sensitivity of the electrophoresis test can be ensured. Therefore, the invention further discloses the application of the compound in nucleic acid gel electrophoresis tests.

Further, the present invention discloses a nucleic acid dye comprising the above compound or a salt thereof, such as a chloride salt, a bromide salt or an iodide salt.

The invention also discloses a method for carrying out nucleic acid gel electrophoresis test by using the nucleic acid dye, which comprises the following steps of firstly preparing the agar gel, then adding the nucleic acid dye, and carrying out electrophoresis test to obtain an electrophoresis picture. Preferably, the addition amount of the nucleic acid dye is 0.01-0.015 micromole of the nucleic acid dye added into 50 mL of agar gel; the compound of the invention is used as nucleic acid dye, and has less dosage, high sensitivity and accurate detection result.

The invention discloses a novel compound, which has stable molecular structure, simple and controllable preparation method, high sensitivity when being used as a nucleic acid dye, and almost no influence on the mobility of DNA, thereby ensuring the true level of DNA migration and reflecting the true test result; when the fluorescent dye is used for gel test, the problem of obvious fluorescence quenching caused by the fact that the existing fluorescent dye can form a dimer in a high-concentration aqueous solution is solved, and the compound provided by the invention has a large amount of carried charges and a large molecular weight, so that the compound cannot penetrate through a cell membrane to enter a human body to cause damage, namely is nontoxic. The compound of the present invention is therefore a highly sensitive, safe and non-toxic nucleic acid dye.

Drawings

FIG. 1 is a molecular weight measurement of Compound 1 of Table 1;

FIG. 2 is a molecular weight measurement of Compound 8 of Table 1;

FIG. 3 is a gel electrophoresis test chart of a conventional product;

FIG. 4 is a gel electrophoresis test chart of the compound of the present invention.

Detailed Description

Example one

Putting 60.0g of 3, 8-diamino-6-phenylphenanthridine, 360ml of DMF (dimethyl formamide) and 37.2ml of Pyridine into a 1000ml three-neck round-bottom flask, mechanically stirring, and cooling to 0-5 ℃ in an ice water bath; dropwise adding 42ml of II, and keeping the temperature at 0-5 ℃ in the dropwise adding process; after the dropwise addition is finished, the reaction is continued for ten hours until the reaction is finished; performing suction filtration, adding the filter cake into about 2L of pure water, mechanically stirring for 30min, and performing suction filtration; washing the filter cake with 2L of pure water again, and performing suction filtration until the filter cake is dry; drying under reduced pressure to constant weight to obtain 48 g of yellow solid; 347g of 6-iodoethyl hexanoate is weighed and placed in a 2000ml three-neck flask, 39.4g of intermediate I is added under mechanical stirring, the mixture is heated to 110 ℃ in an oil bath, and the reaction lasts 3 days until the reaction is finished; cooling to 80 ℃, adding 1500ml EA, refluxing for 1h, stopping heating, and naturally cooling to room temperature; performing suction filtration, and washing a filter cake for 3 times by using ethyl acetate; carrying out decompression and suction drying to constant weight to obtain 36 g of a product intermediate II; cooling 125 ml of oleum to zero, and adding 12 g of the intermediate III into the cooled oleum; slowly heating the mixed solution to room temperature under the condition of stirring, and continuously stirring for 12 hours until the reaction is finished; the mixture was cooled to-20 ℃ and then poured into-10 ℃ sodium iodide solution (250 g of sodium iodide dissolved in 250 ml of water); stirring the mixed solution at about zero ℃ for 1 hour to precipitate a large amount of precipitate, filtering the mixed solution, and drying the precipitated solid to constant weight to obtain 4.6 g; weighing 12.5g I intermediate IV, placing in a 200ml three-necked flask, adding DMF65ml, mechanically stirring, and cooling to 0-10 deg.C; dropwise adding 12 ml of diisopropylethylamine, and keeping the temperature at 0-10 ℃; adding TSTU in batches until the raw materials are completely reacted; calculating the dosage of 2,2 '-oxo-bis (ethylamine) dihydrochloride according to the dosage of TSTU, firstly adding 70-80% of the calculated dosage, and adding about 10ml of diisopropylethylamine before adding 2,2' -oxo-bis (ethylamine) dihydrochloride each time until the reaction is complete; at normal temperature, the DMF and the excess diisopropylethylamine are pumped out under reduced pressure; after completely pumping, adding 1000ml of acetonitrile, stirring and crystallizing overnight; carrying out suction filtration, adding 1000ml of acetonitrile into a filter cake, stirring overnight, and washing once again; suction filtration, filter cake decompression drying to constant weight, 8.3g red black solid.

Example two

In a 100 mL three-necked flask equipped with a reflux condenser, 40 mL of chlorobenzene was added, and SM (10 g) was added thereto with stirring. Dimethyl sulfate (4 mL) was added, the three were mixed, warmed to reflux, followed by TLC, developing solvent: methanol (5%) ethyl acetate (95%) and a small amount of acetic acid was added to the end of the reaction (about 2-3 hours). Adding 10mL of ethanol, continuously refluxing for 15 min, stopping heating, cooling to room temperature, directly filtering to remove the solvent, washing a filter cake with diethyl ether for 2-3 times, and vacuum-drying the solid to obtain a product intermediate I (8 g).

A250 mL three-necked flask equipped with a mechanical stirrer and a reflux condenser was charged with intermediate I (7 g), 100 mL of acetonitrile, and 20 mL of water, and gradually heated to a reflux state, and then reduced iron powder (15 g) and ferric trichloride (200 mg) were added to the reaction system, and the reflux reaction was continued for 2 hours. And (3) putting filter paper and diatomite into the sand core funnel, carrying out hot filtration to remove iron powder and iron salt, cooling the obtained filtrate to room temperature, and separating out solids. The solid was obtained by filtration and dried under vacuum to give intermediate II (5 g).

In a 1L three-necked flask equipped with mechanical stirring and a reflux condenser, ethyl 6-iodohexanoate (30 g) was added, and then intermediate II (4.67 g) was added thereto with stirring. Gradually heating to 110 ℃ for reaction, tracking by TLC, developing agent: chloroform: methanol: ethyl acetate: acetic acid = 15:2:2:1 to the end of the reaction (approximately 3-4 days are required). The temperature is reduced to 80 ℃, and 150 mL ethyl acetate is added for reflux reaction for 1 hour. Cooled to room temperature and stirred overnight. The ethyl acetate and 6-iodoethyl hexanoate were removed by direct filtration, the filter cake was washed 2-3 times with ethyl acetate and the resulting solid was dried in vacuo to give the product intermediate III (4.2 g).

In a 100 mL three-necked flask with a reflux condenser and mechanical stirring, 24 mL of 48% aqueous hydrogen bromide was added, and the starting intermediate III (2.4 g) was slowly added to the flask. After the addition, the mixture was stirred at room temperature for one hour. Is heated to 110^oC, until the TLC detection reaction is finished (about 3-4 hours are needed for the reaction). Developing agent: 10% water 90% acetonitrile. After the reaction was complete, the reaction was cooled to room temperature and placed in a refrigerator at 4 ℃ overnight. Solids precipitated, the aqueous hydrogen bromide solution was removed by filtration, and the filter cake was washed with a small amount of ice water. The solid was lyophilized to give intermediate IV (1.3 g).

A25 mL single vial was charged with intermediate IV (430 mg), DMF (15 mL), Et₃N (1 mL) was added to the ice-water bath and stirred for 30 minutes. TSTU (330 mg) was then added and the reaction stirred further. Until the end of the TLC run (about 0.5-1 hour for the reaction), the developing solvent: methanol: dichloromethane =2: 8. After the reaction, ethylenediamine (30 μ L) and triethylamine (1 mL) were added to gradually return to room temperature for reaction. TLC detection, developing solvent: water acetonitrile =1:9 (Al)₂O₃A plate). After the reaction, DMF and Et were removed under reduced pressure₃Dissolving the remainder with methanol to prepare Al₂O₃Sample of column, eluent: 3% -8% of H₂O/CH₃And (C) CN. Collecting the product from each tube, removing the solvent by rotation, andthe solid obtained was dried under vacuum to obtain the final product (300 mg).

EXAMPLE III

2-amino-4, 4' -dinitrobiphenyl (17.2 g) and 3-cyanobenzoyl chloride (11 g) were charged in a 250 mL three-necked flask equipped with a reflux condenser, followed by 60mL of chlorobenzene, magnetically stirred and gradually warmed to reflux, and reacted for 4 hours. After the reaction is finished, the reaction product is cooled to room temperature, chlorobenzene is directly removed by filtration, the obtained filter cake is recrystallized and purified by acetic acid, and the obtained solid is dried in vacuum to obtain a product intermediate I (24.1 g).

A250 mL single neck flask equipped with a reflux condenser was charged with intermediate I (18 g) and 60mL nitrobenzene, and 6 mL POCl was added with magnetic stirring₃Gradually heating to 200 deg.C for 3 hr, cooling to room temperature, and distilling under reduced pressure to remove nitrobenzene and POCl₃The remaining solid was recrystallized from ethanol and the resulting solid was vacuum dried to give intermediate II (13.2 g).

A500 mL three-necked flask equipped with a mechanical stirrer and a reflux condenser was charged with intermediate II (14 g), 200mL of acetonitrile, and 40 mL of water, and gradually heated to a reflux state, and then reduced iron powder (30 g) and ferric trichloride (400 mg) were added to the reaction system, and the reflux reaction was continued for 2 hours. And (3) putting filter paper and diatomite into the sand core funnel, carrying out hot filtration to remove iron powder and iron salt, cooling the obtained filtrate to room temperature, and separating out solids. The solid was obtained by filtration and dried in vacuo to give intermediate III (8.5 g).

In a 1L three-necked flask equipped with mechanical stirring, 25 mL of DMF was added and intermediate III (6.2 g) was added with stirring (note: DMF was not added first and no lumps appeared and stirring was impossible). Adding pyridine (3.67 mL), mixing the three, cooling to 0-5 ℃ in an ice bath, starting to dropwise add ethyl chloroformate (6.14 mL), wherein the temperature cannot exceed 15 ℃ in the dropwise adding process, tracking by TLC, and developing agent: petroleum ether ethyl acetate =1: 1, to the end of the reaction. Stirring was continued at room temperature for 30 minutes to facilitate adequate reaction. The obtained sticky solid is directly filtered to remove pyridine and DMF, the solvent obtained by filtering is poured into a waste liquid barrel or water to recover the product (note that the recovered product is slightly impure and needs to be separately placed), the filter cake is poured into water, the filter cake is washed for 2-3 times until no pyridine smell exists, the product is dried at room temperature for 1-2 weeks and then is dried in vacuum, and the product intermediate IV (8.2 g) is obtained.

In a 100 mL three-necked flask equipped with a reflux condenser, 40 mL of chlorobenzene was added, and intermediate IV (5 g) was added thereto with stirring. Dimethyl sulfate (2.1 mL) was added, the three were mixed, warmed to reflux, followed by TLC, developing solvent: methanol (5%) ethyl acetate (95%) and a small amount of acetic acid was added to the end of the reaction (about 2-3 hours). Adding 10mL of ethanol, continuously refluxing for 15 min, stopping heating, cooling to room temperature, directly filtering to remove the solvent, washing the filter cake with diethyl ether for 2-3 times, and vacuum drying the solid to obtain a product intermediate V (4.3 g).

In a 100 mL three-necked flask with a reflux condenser and mechanical stirring, 24 mL of 48% aqueous hydrogen bromide was added, and the starting intermediate V (2.4 g) was slowly added to the flask. After the addition, the mixture was stirred at room temperature for one hour. The reaction was heated to 110 ℃ until the TLC detection reaction was complete (approximately 3-4 hours was required for the reaction). Developing agent: 10% water 90% acetonitrile. After the reaction was complete, the reaction was cooled to room temperature and placed in a refrigerator at 4 ℃ overnight. Solids precipitated, the aqueous hydrogen bromide solution was removed by filtration, and the filter cake was washed with a small amount of ice water. The solid was lyophilized to give intermediate VI (1.23 g).

A25 mL single vial was charged with intermediate VI (86 mg), DMF (5 mL), Et₃N (0.2 mL) was added to the ice-water bath and stirred for 30 minutes. TSTU (66 mg) was then added and the reaction stirred further. Until the end of the TLC run (about 0.5-1 hour for the reaction), the developing solvent: methanol: dichloromethane =2: 8. After the reaction, hexamethylenediamine (6. mu.L) and triethylamine (0.2 mL) were added to gradually return to room temperature for reaction. TLC detection, developing solvent: water acetonitrile =1:9 (Al)₂O₃A plate). After the reaction, DMF and Et were removed under reduced pressure₃Dissolving the remainder with methanol to prepare Al₂O₃Sample of column, eluent: 2% -6% of H₂O/CH₃And (C) CN. The product from each tube was collected, the solvent was removed by spinning, and the resulting solid was dried under vacuum to give the final product (54 mg).

Other dye compounds can be prepared by replacing some raw materials according to the preparation method, and the specific structural formula is as follows:

compound 1:

compound 2:

compound 3:

compound 4:

compound 5:

compound 6:

compound 7:

compound 8:

compound 9:

compound 10:

wherein the compounds numbered 7 and 9 were made according to the last step of example one using ethylene diamine and DAPEE (H), respectively₂NCH₂CH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂CH₂NH₂) The compound 8 is formed by directly sulfonating the starting material 3, 8-diamino-6-phenylphenanthridine and then connecting with TDIEEA; compounds 4, 5 and 6 were prepared according to example two, the last step being linked using 2,2' -oxydianeamine dihydrochloride (2, 2-oxydis (ethylene) dichloride), hexamethylenediamine and DAPEE as bridges, respectively; compounds 1, 2 and 10 were prepared according to the last step of example III using DAPMA, DAPEE and ethylenediamine, respectively, as bridges.

The chemical structural formula of TDIEEA is:

the chemical structure of DAPMA is:

FIGS. 1 and 2 are molecular measurements of Compound 1 and Compound 8, respectively, showing correct molecular weights, indicating that the products of the present invention conform to the theoretical design. The molecular weights of the compounds 1 to 10 are 1051.79, 1126387, 1022.76, 1181.01, 1193.06, 1297.17, 969.14, 1072.14, 1143.38 and 966.8 in sequence.

Compared with the existing similar products, the compound of the invention, as a nucleic acid dye, not only maintains the special high sensitivity and is safer and nontoxic, but also solves the tailing phenomenon in nucleic acid electrophoresis in the prior art.

Taking a compound with the following structural formula (a compound numbered as 1 in table 1) and a similar product of the company Biotium in America as an example, performing gel electrophoresis imaging, and referring to the attached drawings 3 and 4, a comparison result shows that the product of the invention not only has equivalent dye brightness, but also can obtain higher dyeing effect compared with the existing similar product; as can be seen from FIG. 3, the electrophoretic mobility of nucleic acids with different amounts is significantly affected by the electrophoresis of the existing product; as shown in FIG. 4, the gel electrophoresis test results obtained under the same conditions using the product of the present invention show that the effect of the product as a dye on the nucleic acid mobility is very small.

The specific experimental conditions were as follows:

gel concentration, prepare 1% agarose gel (e.g., 1%, i.e., 1 g agarose in 100 mL 1 × TBE); nucleic acid dye concentration, agarose solution added 10000 nucleic acid dye, per 50 mL gel added 5 ul nucleic acid dye (2.6 micro mol per mL);

electrophoresis conditions: electrophoresis is carried out for 60 min under the voltage of 100V in electrophoresis buffer solution of 1 × TBE;

the sample loading sequence from left to right is as follows: super DNA Marker (5 ul, 2.5 ul, 1.25 ul), 100bp Ladder (5 ul, 2.5 ul, 1.25 ul), 1kb Ladder (5 ul, 2.5 ul, 1.25 ul), DM2000 (5 ul, 2.5 ul, 1.25 ul), all the samples from Kangji century;

and (4) observing results: the stained gel was observed through a standard transilluminator (302 nm) and photographed at 400ms exposure to obtain an electrophoretogram.

Claims

1. A nucleic acid gel electrophoresis test method comprises the following steps of firstly preparing agar gel, then adding nucleic acid dye, and then carrying out electrophoresis test to obtain an electrophoresis chart; the nucleic acid dye comprises a compound of the following chemical structural formula:

in the compounds, the cationic compound further contains an anion.

2. The method for nucleic acid gel electrophoresis testing according to claim 1, wherein: the anion includes chloride, bromide, or iodide.

3. The method for nucleic acid gel electrophoresis test according to claim 1, wherein the amount of the nucleic acid dye added is 0.01 to 0.015 micromoles per 50 mL of the agar gel.