CN101187633A - Double stranded nucleic acid precision quantifying method - Google Patents
Double stranded nucleic acid precision quantifying method Download PDFInfo
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- CN101187633A CN101187633A CNA2007101798165A CN200710179816A CN101187633A CN 101187633 A CN101187633 A CN 101187633A CN A2007101798165 A CNA2007101798165 A CN A2007101798165A CN 200710179816 A CN200710179816 A CN 200710179816A CN 101187633 A CN101187633 A CN 101187633A
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- evagreen
- double
- mix
- chain nucleotide
- nucleic acid
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Abstract
The invention discloses a method for quantifying precisely double-chain nucleic acid, which comprises following steps: using EvaGreen TM dye solution to mix with testing double-chain nucleotide solution and incubate in the condition of avoiding light and room-temperature, and using fluorescence detector devices as a Modulus spectrophotometer, a fluorescent enzyme labeled instrument, a real-time quantitative PCR instrument and the like to test quantitatively fluorescent strength which is produced after dye is combined with purified double-chain nucleotide. Compared with a traditional ultraviolet testing method of the double-chain nucleic acid, the invention is more rapidly, accurate and stable. Compared with a normal ultraviolet testing method of the double-chain nucleic acid, the invention has strong specificity and high sensibility, and can test quantitatively the double-chain nucleic acid as genome DNA rapidly.
Description
Technical field
The invention belongs to biological technical field, a kind of EvaGreen of utilization that the present invention relates to
TMDyestuff carries out the method for accurate quantification to double-strandednucleic acid.
Technical background
The method of detection by quantitative double-strandednucleic acid such as genomic DNA has spectrophotometric method, detects the light absorption value at 260nm place, dna content=OD
260* 50 μ g/ml, but the method operation more complicated, and error is bigger; Another kind of common method is to utilize ethidium bromide in conjunction with double-strandednucleic acid such as genomic DNA molecule, under ultraviolet excitation, produce red fluorescence, fluorescence intensity is directly proportional with double-strandednucleic acid such as genomic DNA amount, utilize typical curve, can carry out double-strandednucleic acid such as genomic DNA quantitative test, because ethidium bromide is a carcinogen, should not promote.
Present people use SYBR Green I instead and SYBR Gold is the fluorescent quantitation detection double-strandednucleic acid such as the genomic DNA of dyestuff.Because these two kinds of fluorochromes are only with double-strandednucleic acid such as genomic DNA is double-stranded combines, with the dna double chain combination after send fluorescence, fluorescence intensity is directly proportional with dna double chain product amount, can carry out double-strandednucleic acid such as genomic DNA quantitative test according to typical curve.But because general dyestuff instability, high temperature degradation sees that light easily decomposes, radiative spectral width, and light stability is poor, detects the background height, and accuracy is lower, also should not promote.Must seek the fast, efficient, safe detection by quantitative double-strandednucleic acid of a kind of energy such as the method for genomic DNA for this reason.
EvaGreen
TMBe the green fluorescence nucleic acid dye, after dyestuff and double-strandednucleic acid such as genomic DNA combination, it excites and emission spectrum and fluorescein and SYBR
TMGreen I is similar.This makes this dyestuff to be directly used in to have disposed the instrument of any excited by visible light device in 488nm Argon ion laser or this SPECTRAL REGION.EvaGreen
TMGood thermal stability and hydrolytic stability are arranged, for routine operation is provided convenience.EvaGreen
TMItself does not have fluorescence, but and dsDNA in conjunction with after can send the fluorescence of high brightness, this specific character makes it be particularly suitable for conventional double-strandednucleic acid such as genomic DNA concentration is determined.(the Ai Musishi test result of NY) carrying out shows, EvaGreen for LitronLaboratories, Rochester in the independent experiment chamber
TMThere are not mutagenicity and cytotoxicity.Experiment shows, EvaGreen
TMDo not have cell leakage fully, this may observe hypotoxic key factor.In contrast, SYBR
TMGreen I is considered to have mutagenesis efficiently and strengthens characteristic, has suppressed the repair mechanism of normal DNA in the cell probably.SYBR
TMThe toxicity of Green I may to enter the ability of cell fast relevant with it.Yet with EvaGreen
TMIt is still unsound that dyestuff is used for the detection means of double-strandednucleic acid, is difficult to the Color of obtaining in actual applications.
Summary of the invention
The object of the present invention is to provide a kind of simple, fast, high specificity, highly sensitive can adopt EvaGreen
TMThe method of dyestuff Mix detection by quantitative double-strandednucleic acid such as genomic DNA.
The present invention EvaGreen
TMHigh concentration concentrated dye solution (U.S. biotium company, product article No.: 31002) be made into Mix and mix the lucifuge incubated at room with double chain nucleotide solution to be measured, detect its fluorescence intensity,, obtain the concentration of double chain nucleotide solution to be measured by comparing with typical curve.
Wherein, EvaGreen
TMThe preparation program of dye solution Mix is:
Na
2HPO
4 0.005～0.015mol/L
NaH
2PO
4 0.005～0.015mol/L
NaCl 0.1～0.2mol/L
EDTA·2Na 2～3mmol/L
EvaGreen 4.5～5.5μmol/L
Glycerine, 3～8%
Last phosphoric acid is transferred pH to 7.1～7.5
All the other are distilled water.
Described EvaGreen
TMThe preparation preferred version of dye solution Mix is:
0.01mol/L?Na
2HPO
4
0.01mol/L?NaH
2PO
4
0.15mol/L?NaCl
2.5mmol/L?EDTA·2Na
5μmol/L?EvaGreen
5% glycerine,
Last phosphoric acid is transferred pH to 7.3
All the other are distilled water
Can adopt the multi-functional luminosity meter of Modulus, fluorescence microplate reader or real-time quantitative PCR instrument to detect fluorescence signal.Detect wavelength X
Abs/ λ
Em=500/530nm (DNA combination).
Double chain nucleotide of the present invention comprises genomic DNA or its segment, plasmid DNA or its segment and artificial double-stranded sequence.
Use EvaGreen
TMDyestuff Mix can be to the double-stranded DNA direct quantitative of at least 100 μ l samples.The detection sensitivity of the multi-functional luminosity meter of Modulus is the DNA of 50pg in the 100 μ l samples.Even introduced common several pollutants such as salt, urea, ethanol, chloroform, detergent, albumen or agar in the nucleic acids for preparation process, the linearity of this testing result still can obtain.The fluorescence volume that RNA during experiment detects and single stranded DNA produce is minimum, when using EvaGreen
TMFind when dyestuff Mix experimentizes with the multi-functional luminosity meter of Modulus: the single stranded DNA of identical mole number and RNA almost do not have any influence to quantitative result.The present invention also provides a kind of detection method (adopting the multi-functional luminosity of Modulus to count example), and it comprises the steps:
1) double-strandednucleic acid such as genomic DNA standard items and unknown sample are mixed with 50 μ L solution of series concentration, in each solution, add the Mix of 50 μ L EvaGreen, fully mix.
2) on the multi-functional luminosity meter of Modulus, select blue module, survey the fluorescent value of double-strandednucleic acid such as genomic DNA standard substance earlier, set up typical curve.
3) then unknown sample double-strandednucleic acid such as genomic DNA are detected, the multi-functional luminosity meter of Modulus directly draws concentration value.
The advantage of the method for the invention:
1) adopts EvaGreen
TMThe method of dyestuff Mix detection by quantitative double-strandednucleic acid such as genomic DNA is simple to operate, and fast, it quantitatively has adaptability widely to the double-strandednucleic acid such as the genomic DNA of all samples.
2) adopt EvaGreen
TMThe method of dyestuff Mix detection by quantitative double-strandednucleic acid such as genomic DNA has higher sensitivity, can detect the double-strandednucleic acid such as the genomic DNA template of pg level.
3) adopt EvaGreen
TMThe method of dyestuff Mix detection by quantitative double-strandednucleic acid such as genomic DNA is less demanding to detecting instrument, as long as have the conventional equipment of capabilities of fluorescence detection such as the multi-functional luminosity meter of Modulus, fluorescence microplate reader, real-time quantitative PCR instrument and multi-functional gel imaging system etc. all can be realized double-strandednucleic acid and EvaGreen
TMThe purpose of the efficient detection of dyestuff specific bond institute emitting fluorescence.
Description of drawings
Fig. 1 measures calf thymus double-strandednucleic acid concentration sensitivity curve map.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of embodiment 1 double-strandednucleic acid genomic DNA
With the plant is example, adopts the CTAB method to extract genomic DNA, takes by weighing 200mg through pretreated sample, among the 1.5mL or 2mL centrifuge tube of the liquid nitrogen precooling of packing into after fully grinding in liquid nitrogen (the sample that need grind does not directly add).Add 1mL and be chilled to 4 ℃ extract in advance, acutely shake mixing after, leave standstill 5min on ice, the centrifugal 15min of 10000g abandons supernatant under 4 ℃ of conditions.Add 600 μ L and be preheating to 65 ℃ lysate, abundant resuspended precipitation keeps 40min at 65 ℃ of constant temperature, during put upside down mixing 5 times.Under the room temperature condition, the centrifugal 10min of 10000g gets supernatant and goes in another new centrifuge tube.Add 5 μ L RNaseA, 37 ℃ of constant temperature keep 30min.Use equal-volume phenol respectively: chloroform: isoamylol (25: 24: 1), extracting twice repeatedly, uses chloroform/isoamylol (24: 1) extracting more once.Under the room temperature condition, the centrifugal 10min of 10000g gets supernatant and goes in another centrifuge tube.Add 2/3 volume isoamylol, 1/10 volume 3mol/L sodium acetate solution (pH5.6) is placed 2-3h for-20 ℃.Under 4 ℃ of conditions, the centrifugal 15min of 10000g abandons supernatant, with 70% ethanol washing precipitation once, pours out ethanol, dries precipitation.Add 50 μ L TE (pH8.0) dissolution precipitations, gained solution is sample gene group dna solution.
Embodiment 2EvaGreen
TMDyestuff Mix preparation and using method
EvaGreen
TMThe prescription 1 of dyestuff Mix solution is as follows: 0.01mol/L Na
2HPO
40.01mol/L NaH
2PO
40.15mol/L NaCl; 2.5mmol/L EDTA2Na; 5 μ mol/L EvaGreen; 5% glycerine, last phosphoric acid is transferred pH to 7.3.
EvaGreen
TMThe prescription 2 of dyestuff Mix solution is as follows: 0.005mol/L Na
2HPO
40.005mol/L NaH
2PO
40.1mol/L NaCl; 2mmol/L EDTA2Na; 4.5 μ mol/L EvaGreen; 3% glycerine, last phosphoric acid is transferred pH to 7.1.
EvaGreen
TMThe prescription 3 of dyestuff Mix solution is as follows: 0.015mol/L Na
2HPO
40.015mol/L NaH
2PO
40.2mol/L NaCl; 3mmol/L EDTA2Na; 5.5 μ mol/L EvaGreen; 8% glycerine, last phosphoric acid is transferred pH to 7.5.
During experiment, need to use testing sample and EvaGreen
TMDyestuff Mix mixes in 1: 1 ratio.Formulations prepared from solutions need can not be used glassware with plastic ware, because some becomes branch to be adsorbed onto the glassware surface in the reagent.Wrapping up in or place dark place with paper tinsel gold paper bag keeps in Dark Place.
Annotate: solution uses the testing result meeting relatively good after preparation in a few hours.
The preparation of embodiment 3 double-strandednucleic acid DNA standard solution, blank liquid and sample solution
The preparation of standard solution.Calf thymus DNA is commonly used to do typical curve, and preparation concentration is the solution of 2 μ g/ml.The EvaGreen that isopyknic calf thymus DNA storage liquid is joined
TMIn the Mix working fluid, fully mix titer ultimate density 1000ng/ml.
The preparation of blank liquid.The sample buffer that adds equal volume is to EvaGreen
TMIn the Mix working fluid, fully mix.
The preparation of sample DNA solution.The sample that adds equal volume is to EvaGreen
TMIn the Mix working fluid, fully mix.
1) sample is hatched
Forward in the 100 μ l trace detection wares dyestuff and the sample mix liquid that mix to lucifuge incubated at room 2-5min.
Annotate: do not introduce bubble in the transfer process.
2) the multi-functional luminosity meter of calibration Modulus
Count example with the multi-functional luminosity of Modulus, adopt with the two DNA of 300ng/ml calf thymus and calibrate.
Annotate: the single-point calibration degree of accuracy can be higher, with a standard sample optimization system identical or close with test sample concentration, for example, as if test sample concentration at 300ng/ml, be with a 500ng/ml standard DNA sample.Preserve current calibration for using in the future.
3) sample analysis
Sample adds in the 100 μ l trace detection wares, clicks " Measure Fluorescence ", and the sample net result can be presented on the function machine display screen, adopts EvaGreen
TMThe prescription 1 of dyestuff Mix solution, prescription 2, prescription 3 record soybean gene group DNA concentration and are respectively 279ng/ml, 275ng/ml, 271ng/ml.
Preparation calf thymus DNA concentration is the solution of 500ng/ml.The EvaGreen that isopyknic calf thymus DNA storage liquid is joined
TMIn the Mix working fluid, fully mix titer ultimate density 500ng/ml.The employing said method is measured, and finally records concentration (three mean value) and is 499.8ng/ml.
Embodiment 6 sensitivity determinations
Preparation calf thymus DNA concentration is the solution of 4ng/ml.It is the solution of 2ng/ml, 1ng/ml, 0.5ng/ml, 0.25ng/ml that gradient dilution becomes concentration, the EvaGreen that isopyknic calf thymus DNA storage liquid is joined
TMIn the Mix working fluid, fully mix, fixed through the multi-functional luminosity instrumentation of Modulus, obtain Fig. 1 curve, sensitivity can reach 0.5ng/ml (be lower than 0.5ng/ml, reading approaches 0).
Claims (10)
1. EvaGreen
TMDye solution Mix, it contains following component:
Na
2HPO
4 0.005～0.015mol/L
NaH
2PO
4 0.005～0.015mol/L
NaCl 0.1～0.2mol/L
EDTA·2Na 2～3mmol/L
EvaGreen 4.5～5.5μmol/L
Glycerine 3～8%
Transfer pH to 7.1～7.5 with phosphoric acid at last
All the other are distilled water.
2. EvaGreen as claimed in claim 1
TMDye solution Mix, it contains following component:
0.01mol/L?Na
2HPO
4
0.01mol/L?NaH
2PO
4
0.15mol/L?NaCl
2.5mmol/L?EDTA·2Na
4.5～5.5μmol/L?EvaGreen
5% glycerine
Transfer pH to 7.3 with phosphoric acid at last
All the other are distilled water.
3. claim 1 or 2 described EvaGreen
TMThe working fluid of dye solution Mix.
4. double chain nucleotide quantitative detection method, it comprises step: with each described EvaGreen of claim 1～3
TMDye solution Mix mixes the lucifuge incubated at room with double chain nucleotide solution equal-volume to be measured, detects its fluorescence intensity, by comparing with typical curve, obtains the concentration of double chain nucleotide solution to be measured.
5. method as claimed in claim 4 is characterized in that, with claim 1 or 2 described EvaGreen
TMDye solution Mix is as working fluid, with double chain nucleotide solution equal-volume mixing rear wall light incubated at room to be measured.
6. as claim 4 or 5 described methods, it is characterized in that incubation time is 2～5min.
7. as claim 4 or 5 described methods, it is characterized in that, also comprise the sample that described double chain nucleotide solution to be measured is mixed with series concentration, then respectively with EvaGreen
TMDye solution Mix mixes hatches.
8. as claim 4 or 5 described methods, it is characterized in that described typical curve is to be that reference material is set up with the calf thymus DNA.
9. as claim 4 or 5 described methods, it is characterized in that, adopt the multi-functional luminosity meter of Modulus, fluorescence microplate reader or real-time quantitative PCR instrument to detect fluorescence signal.
10. as claim 4 or 5 described methods, it is characterized in that described double chain nucleotide is genomic DNA or its segment, plasmid DNA or its segment or artificial double-stranded sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007101798165A CN100554946C (en) | 2007-12-18 | 2007-12-18 | A kind of method of double stranded nucleic acid precision quantifying |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007101798165A CN100554946C (en) | 2007-12-18 | 2007-12-18 | A kind of method of double stranded nucleic acid precision quantifying |
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| Publication Number | Publication Date |
|---|---|
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| CN100554946C CN100554946C (en) | 2009-10-28 |
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392282B (en) * | 2008-10-13 | 2011-04-27 | 天根生化科技（北京）有限公司 | Mixed solution special for PCR for amplifying high GC content fragment and use thereof |
| CN101724295B (en) * | 2009-12-03 | 2013-03-13 | 中国农业大学 | LE Green mixed dye and applications thereof |
| CN103827320A (en) * | 2011-07-25 | 2014-05-28 | 牛津纳米孔技术有限公司 | Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores |
-
2007
- 2007-12-18 CN CNB2007101798165A patent/CN100554946C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392282B (en) * | 2008-10-13 | 2011-04-27 | 天根生化科技（北京）有限公司 | Mixed solution special for PCR for amplifying high GC content fragment and use thereof |
| CN101724295B (en) * | 2009-12-03 | 2013-03-13 | 中国农业大学 | LE Green mixed dye and applications thereof |
| CN103827320A (en) * | 2011-07-25 | 2014-05-28 | 牛津纳米孔技术有限公司 | Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores |
| CN103827320B (en) * | 2011-07-25 | 2017-04-26 | 牛津纳米孔技术有限公司 | Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores |
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| Publication number | Publication date |
|---|---|
| CN100554946C (en) | 2009-10-28 |
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