CN102586411A - Safe and environment-friendly DNA (Deoxyribose Nucleic Acid) silver staining method for polyacrylamide gel - Google Patents
Safe and environment-friendly DNA (Deoxyribose Nucleic Acid) silver staining method for polyacrylamide gel Download PDFInfo
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- CN102586411A CN102586411A CN201110020754XA CN201110020754A CN102586411A CN 102586411 A CN102586411 A CN 102586411A CN 201110020754X A CN201110020754X A CN 201110020754XA CN 201110020754 A CN201110020754 A CN 201110020754A CN 102586411 A CN102586411 A CN 102586411A
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Abstract
The invention relates to a method for dyeing DNA (Deoxyribose Nucleic Acid) on polyacrylamide gel. The method is a safe and environment-friendly method for dyeing acidic silver ion solution. The method sequentially comprises the steps of fixing, primary washing, impregnating, secondary washing, developing, and developing reaction ending. In addition, the invention further relates to a method for detecting the DNA on the polyacrylamide gel, and a kit for the methods.
Description
Technical field
The invention belongs to technical field of biological, particularly, the present invention relates to the method that reducing sugar develops to DNA dyeing on polyacrylamide gel, its safety and environmental protection, step is simple, the running time is short and highly sensitive.In addition, the test kit etc. that the invention still further relates to the method that detects DNA and be used for these methods
Background of invention
Current, electrophoresis DNA isolation and make DNA band colour developing (visual) on polyacrylamide gel has become one of method the most frequently used in the biological detection.Wherein, make that DNA bar on polyacrylamide gel is visual can to adopt optical dye, visual organic dye and silver-colored visual organic dye, like Socryl Blue BRL
[1], brilliant cresyl blue
[2], Viola crystallina
[3], and nile blue
[4,5]Deng, its susceptibility is low and operate consuming time.Dye etc.Optical dye; Like ethidium bromide (EB), SYBR green and SYBR gold; That this method demonstrates on the one hand is easy and simple to handle, sensitivity is suitable; Have on the other hand to show as them and all need expensive special fluoroscopic examination instrument, some optical dyes these shortcomings that are difficult to overcome such as the health that threatens the operator, many fluoroscopic examination unstable results that have the mutagenesis meeting, greatly limited their use
[6-8]And the isolating proteinic detection of electrophoresis on being widely used in polyacrylamide gel of silver-colored dyeing technique, it has incomparable highly sensitive
[9], also have report silver to dye
[10-12]Can be used for nucleic acid
[13]And LPS
[14]Dyeing.
The silver dyeing technique is according to being divided into two big types with used reagent in the infiltration of argentiferous ion solution.One type is called as silver-colored ammonia staining, and it uses silver-ammonia solution gel that infiltrates, and develops the color with the acidic solution that contains formaldehyde, but its susceptibility is low and operating process is time-consuming
[15]The dyeing of the acid silver ion solution of another kind of usefulness, wherein the gel infiltration is in the weakly acidic Silver Nitrate at pH, then under alkaline condition with the formaldehyde reduction, yet it is carrying out nucleic acid (DNA and RNA) when detecting, or dyes highly sensitive but the operating process more complicated
[16], or operating process is simple but sensitivity is not high
[17-19], but all keep away the health hazards of unavoidable formaldehyde reagent itself to human body.
For this reason; The inventor is through long-term practice research, on the basis of silver nitrate method staining, invented surprisingly a kind of with the DNA argentation of glucose as reductive agent, use the reducing sugar replacement carcinogenic, cause allergy and strong volatile formaldehyde reagent; The row method of going forward side by side whole improvement; Suitably improving on the basis of detection sensitivity, simplify step, only in about 45 minutes running time, just can detect the DNA band (can reach 5pg) of pik (picogram) level.In addition, the inventor has also invented glucose-DNA argentation and has been used for test kit of these methods etc.
Summary of the invention
The object of the present invention is to provide on polyacrylamide gel the painted method of nucleic acid (is representative with DNA), it has brought the improvement of over-all properties on detection sensitivity and convenient degree of operation.In addition, the test kit etc. that the present invention also aims to provide the method that detects DNA and be used for these methods.
Particularly, in first aspect, the object of the present invention is to provide on polyacrylamide gel the painted method of DNA; It is acid silver ions staining; It is characterized in that the silver ion reducing agent in the said method is a reducing sugar, instead of formaldehyde is as the development composition.The method of first aspect present invention is used behind the polyacrylamide gel electrophoresis DNA isolation usually; DNA band on the polyacrylamide gel dyes; Although not preferred, this method also can make DNA be wrapped in use afterwards in the polyacrylamide gel with other modes.In this article, silver nitrate method staining is originally as prior art
[15], it comprises fixing step, for the first time washing step, infiltration step, washing step, development step and color development stopping reactions step for the second time successively.Wherein, development step is with the formaldehyde solution polyacrylamide gel that infiltrates.Because the present invention is with respect to the development step of prior art one acid silver ion solution staining; Use reducing sugar to replace formaldehyde reagent; Wherein, avoided the health hazards of formaldehyde itself, and simplified the method for reducing flow process on the whole the operator with the glucose best results.Preferably in first aspect of the present invention, said method comprises fixing step, washing step, infiltration step, washing step, development step and color development stopping reactions step for the second time for the first time successively.Like this, this method has also kept the detection sensitivity of existing acid silver ion solution staining after reaching above-mentioned advantage.
In this article, " successively " refers to the step that is directed against and carries out by the order that it occurred, and can not exchange the order of carrying out with other steps.Preferably in first aspect of the present invention, said method is successively by fixing step, washing step, infiltration step, washing step, development step and color development stopping reactions step for the second time for the first time.
In this article, " for the first time " is used to distinguish the order of washing step on operating process with " for the second time ", do not constitute the restriction to the implementation of washing step own.In this article, " washing " refers to and uses the solvent cleaning polyacrylamide gel, remains in the reagent on the polyacrylamide gel in order to a step on the flush away.Wherein, preferred solvent is water, saline water or damping fluid, like Tris-HCl damping fluid, PBS damping fluid etc., most preferably is deionized water.Preferably in first aspect of the present invention, in the said method for the first time washing step be to use the water washing polyacrylamide gel.The inventor discovers that washing for the first time has significant lifting for final Color, be necessary, but washing time can be long.Therefore preferably wherein, washing time is more than or equal to 3 minutes, and more preferably 5-30 minute, most preferably be 5 minutes, washing times is three times.Wherein most preferred like this mode has been practiced thrift the running time on the basis that guarantees effectively cleaning significantly.
In this article, " infiltration " refers to silver nitrate solution and soaks polyacrylamide gel, uses so that silver ions infiltrates in the polyacrylamide gel and is attached on the DNA in the gel.Preferably in first aspect of the present invention, the inventor discovers that Silver Nitrate content has remarkable influence for final Color in the time of infiltration and the solution.Therefore in order further to improve Color, improve detection sensitivity and save operation time, preferably the infiltration time is 5-60 minute, more preferably 8-30 minute, most preferably is 5 minutes; The concentration of preferred Silver Nitrate is 0.05%-0.4%, and more preferably 0.1%-0.3% most preferably is 0.2%.In addition, as not particularly pointing out, said " solution " is the aqueous solution in this article, and promptly solvent is a water, and preferably showing wherein contained solute clearly is the whole solutes in the said solution; Also preferably for liquid solute, its percentage concentration refers to concentration of volume percent, and for solid solute, its percentage concentration refers to mass percent concentration.
Preferably in first aspect of the present invention, in the said method for the second time washing step be to use the water washing polyacrylamide gel.Wherein, preferred washing time is 10 seconds-5 minutes, and most preferably washing time is 30 seconds; Preferred washing times is twice; Preferred water wherein is deionized water.
In this article, " colour developing " refers to the basic soln that contains reductive agent and soaks polyacrylamide gel, with so that infiltrate the silver ion reduction Cheng Yin on the DNA in the polyacrylamide gel, thereby shows observable color.Wherein, reductive agent is formaldehyde normally, and alkali is weak base, preferably yellow soda ash.Preferably in first aspect of the present invention; Development step is to soak polyacrylamide gel with the solution that contains reducing sugar (is representative with glucose), sodium hydroxide and boric acid in the said method; Its mesoboric acid and sodium hydroxide are formed alkaline buffer system; In addition, boric acid is through having strengthened its reducing power with the glucose effect.The inventor discovers that each components contents has remarkable influence for final Color in the solution, and especially the reductive action (speed of reduction reaction) of reductive agent such as glucose receives to be influenced by the pH value of solution of alkaline buffer system decision.Therefore, preferred concentration sodium hydroxide is respectively 150-300mM, and more preferably 200-300mM most preferably is 250mM; The concentration of boric acid is respectively 0-200mM, and more preferably 75-150mM most preferably is 100mM.In addition, the content of reductive agent reducing sugar also can remarkably influenced the colour developing result, be representative with glucose, the concentration of preferred glucose is 2%-10%, more preferably 6%-10% most preferably is 8%.The time of colour developing is preferably 8-10 minute by whole color developing effect (combining sensitivity and gel background contrasts factor) decision.
In this article, " color development stopping reaction " refers to polyacrylamide gel is soaked in the coupling reaction stop bath, in order to stop aforesaid colour developing.Usually, the coupling reaction stop bath is for containing acetic acid water solution.Preferably in first aspect of the present invention, the color development stopping reactions step is to soak polyacrylamide gel with sodium ethylene diamine tetracetate solution in the said method.Wherein, preferred 1.5% sodium ethylene diamine tetracetate solution, the preferred time is 5 minutes.
In second aspect; The object of the present invention is to provide the test kit that is used for the said method of first aspect present invention; It comprises infiltration solution, chromophoric solution and the coupling reaction stop bath of packing; The solution that wherein infiltrates is silver nitrate solution, and chromophoric solution is the basic soln that contains reductive agent, and the coupling reaction stop bath is the aqueous solution that contains sodium ethylene diamine tetracetate.Preferred said test kit comprises Silver Nitrate infiltration solution, boronic acid containing and the sodium hydroxide of packing, the chromophoric solution and the sodium ethylene diamine tetracetate solution of glucose.In the more preferably said test kit in each solution the content of solute as first aspect present invention institute is preferred.Although not preferred, said test kit can also comprise the water of packing, like deionized water.
In this article, test kit has the accessible implication of those skilled in the art, and in this article, it comprises container separately, respectively packing (that is packing) different solution.Like this, can not mix between the different solution.Wherein, container is any container that can preserve its stored solution, like vial, plastics pot etc., and container that preferably can its stored solution of prolonged preservation.In addition, the test kit of preferred second aspect present invention also comprises the specification sheets that records the said method of first aspect present invention.Specification sheets can be independently, and like the papery specification sheets, it is placed in the test kit; Specification sheets also can be directly to be printed on the test kit, as being printed on the one or more containers in the test kit.
For the ease of understanding, below will describe in detail the present invention through concrete accompanying drawing and embodiment.What need particularly point out is, specific examples and accompanying drawing only are in order to explain, the Reference numeral that especially is positioned at bracket in claims and the specification sheets only is the understanding when reading for ease, does not constitute limitation of the scope of the invention.Obviously those of ordinary skill in the art can explain according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.In addition, the present invention has quoted open source literature, and these documents also are in order more clearly to describe the present invention, and their full text content is all included the present invention in and carried out reference, just look like they full text in specification sheets of the present invention repeated description the same excessively.
Description of drawings
In the gel photograph in the following accompanying drawing, the applied sample amount of each band of Φ X174DNA/HaeIII molecular weight marker is: swimming lane 1,2500pg; Swimming lane 2,1250pg; Swimming lane 3,640pg; Swimming lane 4,320pg; Swimming lane 5,160pg; Swimming lane 6,80pg; Swimming lane 7,40pg; Swimming lane 8,20pg; Swimming lane 9,10pg; Swimming lane 10,5pg.
Fig. 1 influences figure with different reducing sugars to Color after having shown electrophoresis, (A) glucose-argentation of the present invention, (B) wood sugar-argentation, (C) ribose-argentation.
Fig. 2 has shown the dyeing photo of the method for glucose-argentation of the present invention and other 2 kinds of comparative examples; Wherein, (A) glucose-argentation of the present invention; (B) commercial formaldehyde-argentation of GE Healthcare, (C) SYBR
Gold nucleic acid is gel-colored.
Embodiment
Below describe through concrete embodiment, wherein special material, the step that specifies is well-known to those skilled in the art, as can be referring to " molecular cloning experiment guide " books or laboratory manuals such as (Science Press, 2002).
1, experiment material
Acrylic amide, methyl bisacrylamide (Bis), Tetramethyl Ethylene Diamine (TEMED), ammonium persulphate (APS), Tris, boric acid, glucose, wood sugar, ribose, semi-lactosi, rhamnosyl and lactose are available from Sigma-Aldrich Chemical Co. (St.Louis; MO, the U.S.).Φ X174DNA/HaeIII molecular weight marker (Cat#G1761) is available from Promega Corporation (Madison, WI, the U.S.).DNA silver transfection reagent box (Cat#70-5006-88) is available from GE Healthcare (Uppsala, Sweden).SYBR
the gel-colored test kit of Gold nucleic acid is available from Molecular Probes (Eugene; OR, the U.S.).Other chemical reagent are analytical pure, buy through commercially available channel.
2, electrophoresis and image analysis
Polyacrylamide gel electrophoresis method according to routine is carried out; Concise and to the point process is following: dissolve acrylic amide and aggregate into 8% polyacrylamide gel (80mm * 100mm * 0.75mm) with tbe buffer liquid (pH 8.0 for 89mM TB, 2mM EDTA); Wherein, the amount ratio of acrylic amide and Bis is 29: 1.Φ X174DNA/HaeIII molecular weight marker doubly doubly is diluted to each concentration and goes up appearance to each swimming lane of gel with Tris-EDTA (TE) damping fluid (pH 8.0 for 10mM EDTA, 100mM Tris-Cl): make the applied sample amount of Φ X174DNA/HaeIII molecular weight marker of each swimming lane be respectively 2500; 1250,620,310; 160,80,40; 20,10 and 5pg.Carry out electrophoresis with Miniprotean III dual slab cells (BioRad, Hercules, CA, the U.S.) and PAC 300 (BioRad) with the parameter of 20mA/ gel, electrophoresis liquid is above-mentioned tbe buffer liquid.Electrophoresis arrives the gel bottom margin until tetrabromophenol sulfonphthalein, generally needs 30 minutes.
Gel after electrophoresis is accomplished dyes with following staining respectively and observes the colour developing result.Gel with cma staining utilizes scanner (Epson Perfection V700Photo, the U.S.) to become image with the resolution scan of 600dpi; Take photos (image) with Molecular Imager Gel Doc XR imaging system (BioRad) under the uv lamp of 302nm wavelength shines with SYBR
Gold stained gel, the time shutter is 2.0 to 3.0 seconds.
3, the embodiment of DNA silver nitrate method staining of the present invention
Behind the electrophoresis, use a plurality of gels to carry out a series of experiments that following experiment condition changes: gel washs in the 100mL deionized water, and washing time is respectively 0 minute (not washing), 10 minutes, 30 minutes and washing are spent the night.Then, gel infiltrated respectively 5 to 60 minutes in 50mL contains the solution of Silver Nitrate, and wherein the concentration of Silver Nitrate is respectively 0.05%-0.4%.Take out gel then, washed twice in the 100mL deionized water, each 20 seconds.Take out gel then, immerse the solution colour developing that 50mL contains glucose, sodium hydroxide and boric acid, wherein the concentration of glucose is respectively 2%-10%, and concentration sodium hydroxide is respectively 150-300mM, and the concentration of boric acid is respectively 0-200mM.Treat that color manifests and gel is taken out in stable back (needing 8-10 minute) and immerse the reaction of 50mL1.5% sodium ethylene diamine tetracetate (EDTA-2Na) solution color development stopping.
The result of this serial experiment is following:
(1) behind the electrophoresis fixedly washing time to painted influence
Material in the electrophoresis like Tris, EDTA, boric acid etc., may disturb silver ions and DNA to form mixture, makes the relative DNA band of background overrich.Therefore, the different fixed time can have different influences to final color developing effect.Find through the result; Gel washed in stationary liquid 10 minutes; Pass through deionized water wash then three times, each 5 minutes, be enough to produce good colour developing result; Long washing time can not significantly promote color developing effect, does not fix and wash washing the sensitivity of detection is greatly reduced.
(2) in the infiltration solution component concentration and infiltration time to painted influence
Infiltrate respectively with the concentration of 0.05%-0.4% Silver Nitrate in the infiltration solution, the readability of comprehensive detection sensitivity, picture contrast and background finds that 0.2% silver nitrate concentration is best.Thereby the Silver Nitrate of greater concn will cause the denseer reduction of background contrast gradient, and the Silver Nitrate of lower concentration will cause sensitivity to reduce.In addition; The infiltration time was respectively 5 to 60 minutes; The result finds that the colored intensity of DNA band just can reach maximum strength under 5 minutes situation of infiltration; Prolonging the infiltration time does not more change the result, can't reach maximum sensitivity thereby be less than the intensity that then was not enough to reach maximum in 5 minutes.
(3) in the chromophoric solution each component concentration to painted influence
In the chromophoric solution respectively with the 2%-10% glucose concn; 150-300mM naoh concentration and 0-200mM boric acid concentration develop the color; Take all factors into consideration reduction rate, background depth and sensitivity, find 8% glucose concn, 250mM naoh concentration and 100mM boric acid concentration are best; The glucose of lower concentration will cause developing time long, and background is dirtier; The glucose of greater concn will cause background color to deepen poor contrast.
(4) in the chromophoric solution different reducing sugars to painted influence
Respectively to the investigation that develops the color of wood sugar, semi-lactosi, ribose, rhamnosyl, lactose, glucose, the result shows (table 1) in the chromophoric solution, and the Color of semi-lactosi and lactose is a muting sensitivity, background subtraction; The dyeing background of wood sugar and ribose is dark, and sensitivity is low; The no phenomenon of rhamnosyl dyeing; Comparatively speaking, the coloration result of glucose is better, shows as background clear, highly sensitive (sensitivity than wood sugar and ribose increases 16-18 respectively doubly), in 8-10 minute, can obtain satisfactory result, as best preferred reagent.
The different reducing sugars of table 1. are to painted influence
(5) different stop buffer compositions are to painted influence
Investigate different stop buffer composition HAc
[20], Tris/HAc
[21], EDTA
[22]To the influence of coloration result, the result find the above two can be to some extent reducing sensitivity and band contrast gradient, the 1.5%EDTA-2Na test solution then can not change staining power.
4, the commercial silver nitrate method staining of comparative example 1:GE Healthcare
Manufacturers instruction according to the DNA of GE Healthcare silver transfection reagent box (Cat#70-5006-88) carries out; Primary process is following: gel immersed in the solution that 125mL contains 24% ethanol and 0.6% Phenylsulfonic acid (BSA) fixing 30 minutes behind the electrophoresis; Contained in the solution of 0.2% Silver Nitrate and 0.07%BSA infiltration 30 minutes at 125mL then, washing 1 minute in the 125mL deionized water then.Gel immersed in the solution that 125mL contains 2.5% yellow soda ash, 0.1% formaldehyde and 0.002% Sulfothiorine colour developing 6 minutes then.Then, gel immerses color development stopping reaction in the solution that contains 1%HAc, 5% sodium acetate and 10% glycerine.
5, comparative example 2:SYBR
Gold nucleic acid is gel-colored
Manufacturers instruction according to SYBR
the gel-colored test kit of Gold nucleic acid of Molecular Probes carries out; Primary process is following: with tbe buffer liquid (89mM TB; 2mM EDTA; PH 8.0) with 10; 000 * SYBR
Gold staining fluid be diluted to 1 *; Behind the electrophoresis; Gel with the 100mL deionized water wash once infiltrated 30 minutes at 50mL SYBR
Gold staining fluid then.
6, the result relatively
In acid silver ions dyeing process in the past, formaldehyde as reductive agent have high volatile volatile, carcinogenic, cause supersensitivity, very big to operator's health hazards.The present invention uses reducing sugar and replaces the acid silver ions dyeing process of this DNA of formaldehyde, and it is safety and environmental protection not only, and step is simple, can accomplish in the clock time at about 45 minutes, far is shorter than additive method.This modification method can detect the DNA band to 5pg; The commercial acid silver ions dyeing process and SYBR
the gel-colored method of Gold nucleic acid that in sensitivity, are superior to GE Healthcare, both can only detect the DNA band of 20pg the back.And compare with the commercial acid silver ions dyeing process of GEHealthcare, dyeing time shortened to 45 minutes from about 100 minutes, and whole process is also obviously simplified.
Reference
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[13]Somerville，L.L.，and?Wang，K.，Biochem.Biophys.Rex?Commun.1981，102，53-58.
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Claims (10)
- On polyacrylamide gel to the painted method of DNA, it is acid silver ion solution staining, it is characterized in that, uses reducing sugar, is representative with glucose, replaces formaldehyde as silver ion reducing agent.
- 2. the described method of claim 1, it comprises fixing step, washing step, infiltration step, washing step, development step and color development stopping reactions step for the second time for the first time successively.
- 3. the described method of claim 2, wherein washing step is use the water washing polyacrylamide gel for the first time, preferably washing time more preferably 5-30 minute, most preferably was 5 minutes more than or equal to 3 minutes; Washing times is 1-6 time, more preferably 2-4 time, most preferably is 3 times.
- 4. the described method of claim 2, the step of wherein infiltrating are with containing silver nitrate solution infiltration polyacrylamide gel, and preferably the infiltration time is 2-30 minute, more preferably 5-10 minute, most preferably is 5 minutes.
- 5. the described method of claim 2, wherein washing step is to use the water washing polyacrylamide gel for the second time.
- 6. the described method of claim 2; Wherein development step is with containing reducing sugar (is representative with glucose), sodium hydroxide and BAS infiltration polyacrylamide gel; The concentration of preferred glucose is 2%-10%, and more preferably 6%-10% most preferably is 8%; Preferred concentration sodium hydroxide is 150-300mM, and more preferably 200-300mM most preferably is 250mM; The concentration of boric acid is 0-200mM, and more preferably 75-150mM most preferably is 100mM.
- 7. the described method of claim 2, wherein the color development stopping reactions step is with sodium ethylene diamine tetracetate solution infiltration polyacrylamide gel.
- 8. the test kit that is used for arbitrary said method of claim 1-7, it comprises the chromophoric solution that contains silver nitrate solution, sodium hydroxide solution, BAS and the sodium ethylene diamine tetracetate solution of packing.
- 9. on polyacrylamide gel, detect the method for DNA, it is included in and carries out electrophoresis on the polyacrylamide gel, carries out the arbitrary said method of claim 1-7 then.
- 10. the test kit that is used for the said method of claim 9, it comprises the polyacrylamide gel electrophoresis reagent of packing, the chromophoric solution that contains silver nitrate solution, sodium hydroxide solution, BAS and sodium ethylene diamine tetracetate solution.
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Cited By (2)
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| CN106596232A (en) * | 2016-12-13 | 2017-04-26 | 广州大学 | Silver staining kit for detecting DNA in polyacrylamide gel and application of silver staining kit |
| CN109212006A (en) * | 2018-08-24 | 2019-01-15 | 北京艾普希隆生物科技有限公司 | A kind of unicellular agarose gel electrophoresis kit |
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| CN101703404A (en) * | 2009-08-28 | 2010-05-12 | 东北师范大学 | Method for showing fingerprints on various object surfaces and keeping DNA information |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596232A (en) * | 2016-12-13 | 2017-04-26 | 广州大学 | Silver staining kit for detecting DNA in polyacrylamide gel and application of silver staining kit |
| CN106596232B (en) * | 2016-12-13 | 2019-04-30 | 广州大学 | The silver staining kit of DNA and its application in a kind of detection polyacrylamide gel |
| CN109212006A (en) * | 2018-08-24 | 2019-01-15 | 北京艾普希隆生物科技有限公司 | A kind of unicellular agarose gel electrophoresis kit |
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Application publication date: 20120718 |