CN105695571A - DNA quantitative method based on rolling circle amplification - Google Patents
DNA quantitative method based on rolling circle amplification Download PDFInfo
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- CN105695571A CN105695571A CN201610063954.6A CN201610063954A CN105695571A CN 105695571 A CN105695571 A CN 105695571A CN 201610063954 A CN201610063954 A CN 201610063954A CN 105695571 A CN105695571 A CN 105695571A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a DNA quantitative method based on rolling circle amplification. The method comprises the following steps: performing rolling circle amplification reaction, restriction enzyme digestion reaction and electrophoresis on DNS standard solutions with different concentrations, and quantitatively analyzing the brightness of an objective strap to obtain a brightness value of the objective strap, thereby acquiring a standard curve between the concentration of the DNA standard solution and the brightness value of the objective strap; and then substituting the brightness value of the objective strap corresponding to the to-be-quantified DNA solution into a standard curve formula, thereby finishing the quantification of the to be quantified DNA. Through the adoption of the method disclosed by the invention, the sensitivity is close to the PicoGreen fluorescent dye quantitative method, an expensive device is unnecessary, and only a water bath pot, an electrophoresis apparatus and a plastic blooming instrument are required. The method disclosed by the invention can be used for quantitatively testing chain-typed DNA in a pg level.
Description
Technical field
The present invention relates to the quantitative approach of a kind of DNA, be specifically related to the quantitative approach of a kind of DNA based on rolling circle amplification。
Background technology
DNA is quantitatively for biological study, and food and medicine industry is all critically important。A lot of diverse ways are all used for the quantitative of DNA:
The most classical and most common method is UV spectrophotometry, the party's ratio juris is sample is corresponding at the absorbance of 260nm with the nucleic acid concentration in sample, up-to-date spectrophotometer has only to the DNA solution of 1 microlitre, and cuvette need not be used to test。Spectrophotometry is convenient and uses extensively, but there are some defects: it cannot distinguish between double-stranded DNA, single stranded DNA and free nucleotide, albumen, polysaccharide, phenol and other organic and inorganic matter also can affect the light absorption value at 260nm place, the most important thing is, DNA concentration reaches 5ng/ μ l(5 μ g/ml) time sensitivity will decline, minimum measuring range is 2ng/ μ l。
The sensitiveest quantitative approach is digital pcr, and it is it appeared that the molecule of single DNA。Digital pcr is by sample dispersion to multiple independent real-time quantitative fluorescence (real-time) PCR reactions, and the reaction containing target molecule is positive, and what do not contain is exactly negative；After pcr analysis, it is not necessary to standard curve or internal reference just can calculate the quantity of target molecule in sample。Principle quantitative for RealtimePCR is DNA and the PCR primer of exponential phase of input is corresponding relation；But it is no matter digital pcr or RealtimePCR is required for sequence-specific primer or probe, and needs the experimental facilities of costliness。
The sensitivity of fluorimetry and cost are between spectrophotometry and digital pcr。Fluorescent dye can in conjunction with the DNA in sample, and under the exciting of UV, the dyestuff of the intercalation of DNA can launch fluorescence, thus calculating the concentration of DNA。It is quantitative that many DNA dyestuffs are used for DNA, such as ethidium bromide, Picogreen fluorescent dye。Picogreen can optionally in conjunction with double-stranded DNA, be widely used double-stranded DNA quantitative in, because the Picogreen being not bound with DNA does not have fluorescence, the fluorescent quantitation method sensitivity utilizing picogreen can reach 0.1pg/ul, the 200ul sample at least containing 20pgDNA is needed to be measured, because picogreen is very low to the adhesion of single stranded DNA, therefore RNA, single stranded DNA and the free nucleotide impact on measuring are only small；And picogreen is not quantitatively by the interference of the impurity such as salt, ethanol and chloroform。But fluorescent quantitation needs special instrument, and such as microplate reader and Qubit, not every laboratory all possesses such condition, which has limited the application of the method。
Summary of the invention
It is an object of the invention to provide the quantitative approach of a kind of DNA based on rolling circle amplification, the method is highly sensitive, it is not necessary to expensive equipment, and sample requirement is few。
The general thought of the present invention is, the DNA standard solution adopting variable concentrations carries out quantitative analysis through rolling circle amplification reaction, restriction endonuclease reaction, electrophoresis the brightness to purpose band and obtains the brightness value of purpose band, thus the standard curve obtained between the concentration of DNA standard solution and the brightness value of purpose band；Then the brightness value treating purpose band that quantitative DNA solution is corresponding is substituted into standard curve formula, thus completing to treat that quantitative DNA's is quantitative。
For reaching above-mentioned purpose, the present invention adopts the following technical scheme that
A kind of DNA quantitative approach based on rolling circle amplification, comprises the following steps:
(1) the DNA standard solution of variable concentrations is obtained the corresponding relation between concentration and the brightness value of purpose band of DNA solution respectively through rolling circle amplification reaction, restriction endonuclease reaction, electrophoretic analysis, thus the relation curve prepared between the concentration of DNA solution and electrophoresis purpose band brightness value；
(2) unknown concentration treated that quantitative DNA solution obtains the brightness of purpose band through rolling circle amplification reaction, restriction endonuclease reaction, electrophoretic analysis, bring into the brightness of purpose band the relation curve of step (1) obtains and treat the concentration of quantitative DNA solution；Thus completing to treat that quantitative DNA's is quantitative；
Technological parameter and reagent that rolling circle amplification reaction in described step (2), restriction endonuclease reaction and electrophoretic analysis adopt are consistent with step (1)；
Described step (1) and step (2) operation repetitive simultaneously；
Described rolling circle amplification is: joined by DNA solution in rolling circle amplification sample buffer, within 1～5 minute, obtains mixed liquor in 90～98 DEG C of reactions, is then centrifuged mixed liquor moment；Add reaction buffer, react 1～5 hour in 25～35 DEG C；Last 60～70 DEG C of reactions 8～15 minutes, obtain rolling circle amplification product；
Described restriction endonuclease reaction is: within 0.5～5 hour, obtain the digestion products of rolling circle amplification product with enzyme action 10xbuffer, water and restricted enzyme after being mixed by above-mentioned rolling circle amplification product in 35～40 DEG C of reactions；
Described electrophoretic analysis is: above-mentioned digestion products carries out agarose gel electrophoresis test, and the brightness of purpose band is carried out quantitative analysis。
In technique scheme, in step (1), treat that the volume ratio of quantitative DNA solution, sample buffer and reaction buffer is 1: 2: 2；In step (2), the volume ratio of rolling circle amplification product, enzyme action 10xbuffer, water and restricted enzyme is 10: 6: 21: 3；In electrophoresis test, during electrophoresis reaction, the concentration of agarose gel is 0.6%, utilizes ImageJ software that the brightness of purpose band is carried out quantitative analysis。
In preferred technical scheme, in rolling circle amplification, within 3 minutes, obtain mixed liquor in 95 DEG C of reactions；After adding reaction buffer, react 1～5 hour in 30 DEG C；Last 65 DEG C of reactions 10 minutes, obtain rolling circle amplification product。In restriction endonuclease reaction, within 1 hour, obtain digestion products in 37 DEG C of reactions。Under this reaction condition, adopt the method for the present invention can obtain amplified production accurately and efficiently, be beneficial to the quantitative accuracy of DNA。
The DNA standard solution of variable concentrations refers to the DNA solution of concentration known, and its DNA contained is consistent with treating quantitative DNA；The DNA solution of variable concentrations is at least the DNA solution of 4 kinds of concentration, it is possible to obtain the relation curve between concentration and the brightness value of purpose band of DNA solution accurately。
Above-mentioned DNA quantitative approach is properly termed as RCA quantitative method, and its sensitivity can reach 10pg/ μ L, and scope is from 10pg/ μ L to 242ng/ μ L；Sample requirement is few, it is only necessary to 1-2 μ L can be carried out measuring, and does not need the instrument of any costliness；Adopt technique scheme that the shape size of DNA is not particularly limited, be suitable for the double-stranded cyclic DNA of 1-5kb size, can be applied equally to great majority < the single stranded circle DNA of 10kb, and can be applicable to linear DNA。
In the present invention, the solvent of dissolving DNA is prior art, such as distilled water, Tris-Cl；Restricted enzyme is prior art, and those skilled in the art can select voluntarily according to the kind treating quantitative DNA；Moment centrifugal treating is the one of centrifugal treating；Currently preferred sample buffer includes random primer, Tris HCl (400mM, pH8.0), KCl (160mM)；Reaction buffer includes Tris-HCl (125mM, pH7.5), MgCl2(25mM)、(NH4)2SO4(25mM), DTT (10mM), dNTP (using final concentration of 0.2mM)；Enzyme action 10xbuffer is buffer, supports the use with restricted enzyme。In the present invention, the reaction condition that unknown DNA solution is quantitative, all reacts the same with the DNA preparing standard curve including technological parameter, interpolation reagent etc.；In order to increase the accuracy of quantitative result further, operation when operation when standard curve is prepared in restriction of the present invention is quantitative with unknown concentration DNA solution is simultaneously parallel to be carried out。DNA standard solution according to known variable concentrations carries out the brightness value of the purpose band of quantitative analysis acquisition through rolling circle amplification reaction, restriction endonuclease reaction, electrophoresis test the brightness to purpose band, obtain the relation between the concentration of DNA solution and the brightness value of purpose band, prepare standard curve；The brightness value of the purpose band that the solution of the DNA of the same race according to unknown concentration records, combined standard curve, complete the quantitative of DNA。
Compared with prior art, present invention have the advantage that
(1) quantitative approach of DNA disclosed by the invention is highly sensitive, and the concentration range of test object is wide, from 10pg/ μ L to 242ng/ μ L；Sample requirement is few, it is only necessary to 1-2 μ L can be carried out measuring；Detection limit is low, it is possible to reach 10pg/ μ L, result is accurate, and error is low, it is to avoid along with testing sample concentration is low in prior art, the shortcoming that sensitivity declines。
(2) shape size of DNA is not particularly limited by method disclosed by the invention, is suitable for the double-stranded cyclic DNA of 2-5kb size, can be applied equally to great majority < double-stranded cyclic DNA of 10kb or single stranded circle DNA, and can be applicable to linear DNA；Applied range。
(3) method device therefor disclosed by the invention is simple, it is not required that complicated reagent, it is to avoid the shortcoming that prior art application is narrow, is suitable for industrial applications。
Accompanying drawing explanation
Fig. 1 is the logarithmic relationship figure between the brightness of purpose band in embodiment one and pGEX4T-1 solution concentration；
Fig. 2 is the electrophoretogram of digestion products in embodiment one；
Fig. 3 is the logarithmic relationship figure between the brightness of purpose band in embodiment two and pUC19 solution concentration；
Fig. 4 is the electrophoretogram of digestion products in embodiment two。
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the invention will be further described
In the present embodiment, composition sample buffer and the source chemicals of reaction buffer, enzyme action system are all existing products, commercially available obtain。
Embodiment one pGEX4T-1(4969bp) RCA quantitative
Prepare 150 μ L sample buffer and 150 μ L reaction buffers；Sample buffer includes random primer, Tris HCl (400mM, pH8.0), KCl (160mM)；Reaction buffer includes Tris-HCl (125mM, pH7.5), MgCl2(25mM)、(NH4)2SO4(25mM), DTT (10mM), dNTP (using final concentration of 0.2mM)。
Sample buffer is separately added in 35 holes, every hole 4 μ L sample buffer；Then respectively by the double steaming solution 2 μ L of pGEX4T-1 that mass concentration is 0pg/ μ L, 10pg/ μ L, 30pg/ μ L, 100pg/ μ L, 1000pg/ μ L, 3000pg/ μ L, each five groups join in 4 μ L sample buffer, pGEX4T-1 double steaming solution (being called U1) the 2 μ L of five groups of 300pg/ μ L is joined in 4 μ L sample buffer simultaneously, carry out rolling circle amplification simultaneously, reacting 3 minutes in 95 DEG C, moment is centrifuged；Again reaction buffer is separately added in 35 holes, every hole 4 μ L reaction buffer, reacts 3 hours in 30 DEG C simultaneously；Last 65 DEG C of reactions 10 minutes, obtain 35 groups of rolling circle amplification product；Within 1 hour, digestion products is obtained in 37 DEG C of reactions after being mixed with 10xbuffer, water and BamHI by above-mentioned rolling circle amplification product。Above-mentioned 35 groups of digestion products are carried out electrophoresis test, and wherein the concentration of agarose gel is 0.6%；And the brightness of purpose band is carried out ImageJ software quantitative analysis:
For the accuracy of test result, every kind of concentration DNA solution preparation of samples five groups, draw, according to the brightness of the purpose band of ImageJ software analysis, the brightness value often organizing result, take its meansigma methods brightness value as concrete concentration pGEX4T-1 double steaming solution；Standard curve is made according to the relation between brightness value and pGEX4T-1 double steaming solution concentration；Seeing accompanying drawing 1, it can be seen that the inventive method highly sensitive, detection limit is low, it is possible to reach 30pg/ μ L。
Accuracy for test result, U1 adopts five groups, the brightness of the purpose band according to ImageJ software analysis show that the brightness value of each result is 2243.255,2272.548,2075.962,1896.184,2217.669, show that the average brightness of purpose band is 2141.124。Brightness value is brought into above-mentioned standard curve, and the concentration obtaining the double steaming solution of pGEX4T-1 is 292.9508pg/uL, error-7.0492pg/ul。
Accompanying drawing 2 is the electrophoretogram of above-mentioned each group of digestion products。As can be seen from the figure 5 bands of each concentration are parallel, and the brightness of purpose band is carried out the brightness value that ImageJ software quantitative analysis draws the double steaming solution of variable concentrations pGEX4T-1。
Embodiment two pUC19(2686bp) RCA quantitative
Prepare 160 μ L sample buffer and 160 μ L reaction buffers；Sample buffer includes random primer, Tris HCl (400mM, pH8.0), KCl (160mM)；Reaction buffer includes Tris-HCl (125mM, pH7.5), MgCl2(25mM)、(NH4)2SO4(25mM), DTT (10mM), dNTP (using final concentration of 0.2mM)。
Sample buffer is separately added in 32 holes, every hole 4 μ L sample buffer；Then respectively by the double steaming solution 2 μ L of pUC19 that mass concentration is 0pg/ μ L, 3pg/ μ L, 10pg/ μ L, 30pg/ μ L, 100pg/ μ L, 1000pg/ μ L, 3000pg/ μ L, each four groups join in 4 μ L sample buffer, double steaming solution (being called U2) the 2 μ L of the pUC19 of four groups of 320pg/ μ L is joined in 4 μ L sample buffer simultaneously, carry out rolling circle amplification simultaneously, reacting 3 minutes in 95 DEG C, moment is centrifuged；Again reaction buffer is separately added in 32 holes, every hole 4 μ L reaction buffer, reacts 3 hours in 30 DEG C simultaneously；Last 65 DEG C of reactions 10 minutes, obtain 32 groups of rolling circle amplification product；Within 1 hour, digestion products is obtained in 37 DEG C of reactions after being mixed with 10xbuffer, water and BamHI by above-mentioned rolling circle amplification product。Above-mentioned 32 groups of digestion products are carried out electrophoresis test simultaneously, and wherein the concentration of agarose gel is 0.6%；And the brightness of purpose band is carried out ImageJ software quantitative analysis。
For the accuracy of test result, the present embodiment every kind concentration samples selects four groups, draws, according to the brightness of the purpose band of ImageJ software analysis, the brightness value often organizing electrophoresis result, takes its meansigma methods brightness value as concrete concentration pUC19 double steaming solution；Standard curve is made according to the relation between brightness value and pUC19 double steaming solution concentration；Seeing accompanying drawing 3, it can be seen that the inventive method highly sensitive, detection limit is low, it is possible to reach 30pg/ μ L。
For the accuracy of test result, U2 adopts four groups, show that according to the brightness of the purpose band of ImageJ software analysis the brightness value of each result is 5598.154, and 4949.962,5670.74,4774.083, show after average that the brightness value of purpose band is 5248.235；Brightness value is brought into above-mentioned standard curve, and the concentration obtaining U2 is 331.0623, error 11.0623pg/ul。
Accompanying drawing 4 is the electrophoretogram of above-mentioned each group of digestion products。As can be seen from the figure 4 bands of each concentration are parallel, and the brightness of purpose band is carried out the brightness value that ImageJ software quantitative analysis draws the double steaming solution of variable concentrations pUC19。
Claims (10)
1. the DNA quantitative approach based on rolling circle amplification, it is characterised in that comprise the following steps:
(1) the DNA standard solution of variable concentrations is obtained the corresponding relation between concentration and the brightness value of purpose band of DNA solution respectively through rolling circle amplification reaction, restriction endonuclease reaction, electrophoretic analysis, thus the relation curve prepared between the concentration of DNA solution and electrophoresis purpose band brightness value；
(2) unknown concentration treated that quantitative DNA solution obtains the brightness of purpose band through rolling circle amplification reaction, restriction endonuclease reaction, electrophoretic analysis, bring into the brightness of purpose band the relation curve of step (1) obtains and treat the concentration of quantitative DNA solution；Thus completing to treat that quantitative DNA's is quantitative；
Technological parameter and reagent that rolling circle amplification reaction in described step (2), restriction endonuclease reaction and electrophoretic analysis adopt are consistent with step (1)；
Described step (1) and step (2) operation repetitive simultaneously；
Described rolling circle amplification is: joined by DNA solution in rolling circle amplification sample buffer, within 1～5 minute, obtains mixed liquor in 90～98 DEG C of reactions, is then centrifuged mixed liquor moment；Add reaction buffer, react 1～5 hour in 25～35 DEG C；Last 60～70 DEG C of reactions 8～15 minutes, obtain rolling circle amplification product；
Described restriction endonuclease reaction is: within 0.5～5 hour, obtain the digestion products of rolling circle amplification product with enzyme action 10xbuffer, water and restricted enzyme after being mixed by above-mentioned rolling circle amplification product in 35～40 DEG C of reactions；
Described electrophoretic analysis is: above-mentioned digestion products carries out agarose gel electrophoresis test, and the brightness of purpose band is carried out quantitative analysis。
2. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: the volume ratio treating quantitative DNA solution, sample buffer and reaction buffer is 1: 2: 2。
3. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: treat that in quantitative DNA solution, solvent is distilled water or Tris-Cl。
4. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: in rolling circle amplification, within 3 minutes, obtain mixed liquor in 95 DEG C of reactions；After adding reaction buffer, react 1～5 hour in 30 DEG C；Last 65 DEG C of reactions 10 minutes, obtain rolling circle amplification product。
5. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: described rolling circle amplification sample buffer includes random primer, Tris HCl, KCl；Reaction buffer includes Tris-HCl, MgCl2、(NH4)2SO4、DTT、dNTP。
6. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: in restriction endonuclease reaction, the volume ratio of rolling circle amplification product, enzyme action 10xbuffer, water and restricted enzyme is 10: 6: 21: 3。
7. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: in restriction endonuclease reaction, within 1 hour, obtain digestion products in 37 DEG C of reactions。
8. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: during electrophoresis test, the concentration of agarose gel is 0.6%；Utilize ImageJ software that the brightness of purpose band is carried out quantitative analysis。
9. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: described in treat that quantitative DNA's is sized to 1～10kb。
10. according to claim 1 based on the DNA quantitative approach of rolling circle amplification, it is characterised in that: described in treat that quantitative DNA is double-stranded cyclic DNA, single stranded circle DNA or linear DNA。
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Application publication date: 20160622 |