CN101724295A - LE Green mixed dye and applications thereof - Google Patents
LE Green mixed dye and applications thereof Download PDFInfo
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- CN101724295A CN101724295A CN200910241487A CN200910241487A CN101724295A CN 101724295 A CN101724295 A CN 101724295A CN 200910241487 A CN200910241487 A CN 200910241487A CN 200910241487 A CN200910241487 A CN 200910241487A CN 101724295 A CN101724295 A CN 101724295A
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Abstract
The invention provides LE Green mixed dye and applications thereof in double-stranded nucleic acid detection. The nucleic acid dye is prepared by comprising LC Green, Eva Green and proper diluent. The double-stranded nucleic acid template (genome DNA, plasmid DNA and the like) and a fluorescent dye LE Green Mix with proper concentration are mixed, and fluorescent detection instruments, such as a Modulus spectrophotometer, a fluorescent microplate reader, a real-time quantitive PCR instrument and the like, are used for quantitively detecting the fluorescence intensity generated after the dye and the purified double-stranded nucleic acid. Compared with the traditional double-stranded nucleic acid ultraviolet detection method, the invention is more rapid, accurate and stable. Compared with the common double-stranded nucleic acid fluorescent detection method, the invention has the advantages of strong specificity and high sensitivity, and can quickly quantitively detect double-stranded nucleic acids, such as genome DNA.
Description
Technical field
The present invention relates to the double chain nucleotide detection technique, be specifically related to a kind of LE Green mixing colouring agent, the invention still further relates to the method that adopts LE Green dyestuff Mix detection by quantitative DNA.
Background technology
The method of detection by quantitative DNA has spectrophotometry at present, detects the light absorption value at 260nm place, dna content=OD260 * 50 μ g/ml, but this method operation more complicated, and also error is bigger; Another kind of common method is to utilize ethidium bromide in conjunction with dna molecular, produces red fluorescence under ultraviolet excitation, and fluorescence intensity is directly proportional with the DNA amount, utilizes typical curve, can because ethidium bromide is a carcinogenic substance, should not promote carrying out the DNA quantitative analysis.
Present people use SYBR Green I instead and SYBR Gold is the fluorescent quantitation detection DNA of dyestuff.Since these two kinds of fluorescence dyes only with the dna double chain combination, with the dna double chain combination after send fluorescence, fluorescence intensity is directly proportional with dna double chain product amount, can carry out the DNA quantitative analysis 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 method for the fast, efficient, safe detection by quantitative DNA of a kind of energy for this reason.
LC Green is the green fluorescence nucleic acid dye, excitation wavelength is 440～470nm, emission wavelength is 470～520nm, LC Green fluorescence dye specific with the dna double chain combination after, can produce intensive fluorescence under the excitation wavelength effect, the DNA concentration that this specific character makes it be particularly suitable for routine is determined.In experimentation, the concentration that need add seldom can reduce experimental cost.Simultaneously, LC Green dyestuff combines the back to the no any restraining effect of PCR reaction with DNA, but other dyestuff has restraining effect as SYBR Green I to PCR, so subsequent experimental is had certain influence.LC Green be difficult for to decompose under hot conditions, good thermal stability, and combine with DNA and to stablize, for routine operation is provided convenience.
Eva Green also is a kind of green fluorescence nucleic acid dye, and after dyestuff and DNA combination, it excites similar to fluorescein and LC Green with emmission spectrum.This makes these two kinds of dyestuffs directly to adopt to have disposed the instrument detecting of any excited by visible light device in 488nm Argon ion laser or this SPECTRAL REGION.Eva Green has good thermostability and stability to hydrolysis; Itself does not have fluorescence, but and double-stranded DNA in conjunction with after can send the fluorescence of high brightness, this specific character makes it be very suitable for determining of conventional DNA concentration.(the Ai Musishi test result of NY) carrying out shows that Eva Green does not have mutagenicity and cytotoxicity for Litron Laboratories, Rochester in Litron independent experiment chamber.Experiment shows that Eva Green does not have cell leakage fully, and this may be to observe hypotoxic key factor.Yet the detection means that Eva Green dyestuff is used for double-strandednucleic acid is still unsound, is difficult to the Color of obtaining in actual applications.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.
Summary of the invention
The objective of the invention is at above-mentioned deficiency provide a kind of simple, fast, high specificity, highly sensitive nucleic acid dye, and the method that adopts this dyestuff detection by quantitative double-strandednucleic acid such as genomic dna.
The present invention is diluted to LE Green dyestuff Mix in proportion with LC Green and Eva Green and utilizes LE Green to combine with double-stranded DNA to produce fluorescence, and intensity of fluorescence becomes positive correlation to come detection by quantitative double-strandednucleic acid such as genomic dna with the amount of DNA, by LEGreen dyestuff Mix is mixed the lucifuge incubated at room with double chain nucleotide solution to be measured, detect its fluorescence intensity, by comparing, obtain the concentration of double chain nucleotide solution to be measured with typical curve.
Wherein, the preparation program of LE Green dye solution Mix is:
Na
2HPO
4 0.01～0.03mol/L
NaH
2PO
4 0.01～0.03mol/L
NaCl 0.15～0.25mol/L
EDTA·2Na 1.5～3.5mmol/L
PVP 0.05-0.20％
TritonX-100 0.05-0.15％
Eva?Green 2.0～4.0μmol/L
LC?Green 1.0～3.0μmol/L
Transfer pH to 7.0～7.5 with phosphoric acid at last
All the other are distilled water.
The preparation preferred version of described LE Green dye solution Mix is:
Na
2HPO
4 0.02mol/L
NaH
2PO
4 0.02mol/L
NaCl 0.20mol/L
EDTA·2Na 2.5mmol/L
PVP 0.1％
TritonX-1000.1％
Eva?Green 3.0μmol/L
LC?Green 2.0μmol/L
Transfer pH to 7.3 with phosphoric acid at last
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 fluorescent signal.
Double chain nucleotide of the present invention comprises genomic dna or its segment, plasmid DNA or its segment and artificial double-stranded sequence.
Count example with the multi-functional luminosity of Modulus, utilize LE Green mixing colouring agent that double-strandednucleic acid is carried out accurate quantification, specifically can comprise the steps:
1) DNA standard substance and unknown sample are mixed with 50 μ L solution of series concentration, in each solution, add 50 μ L LE Green Mix, thorough mixing.
2) on the multi-functional luminosity meter of Modulus, select blue module, survey the fluorescent value of DNA reference material earlier, set up typical curve.
3) then unknown sample DNA is detected, Modulus directly draws concentration value.
With LC Green and Eva Green be mixed in proportion dilution for LE Green mixing colouring agent can be in the blue module of Modulus the quantitative concentration of double-strandednucleic acid, two kinds of dyestuffs can be had complementary advantages, and using LE Green dyestuff 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 pollutents such as salt, urea, ethanol, chloroform, stain remover, albumen or agar in the nucleic acids for preparation process, the linearity of this detected result still can obtain.The fluorescence volume that RNA during experiment detects and single stranded DNA produce is minimum, and find when experimentizing with the multi-functional luminosity meter of LEGreen dyestuff Mix and Modulus: what influence the single stranded DNA of identical mole number and RNA almost do not have to quantitative result.
The advantage of the method for the invention:
1) adopt the method for LE Green dyestuff Mix detection by quantitative DNA simple to operate, fast.It quantitatively has adaptability widely to the DNA of all samples.
2) adopt the method for LE Green dyestuff Mix detection by quantitative DNA to have higher sensitivity, can detect the dna profiling of pg level.
3) the method operation of employing LE Green dyestuff Mix detection by quantitative DNA quantitatively has adaptability widely to the DNA of all samples.Even introduced common several pollutents such as salt, urea, ethanol, chloroform, stain remover, albumen or agar in the nucleic acids for preparation process, the linearity of this detected result still can obtain.
Description of drawings:
Fig. 1 calf thymus DNA concentration and fluorescent value canonical plotting.
Fig. 2 calf thymus double-strandednucleic acid concentration sensitivity graphic representation.
Specific embodiments
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 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 liquor 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 liquor and goes in another new centrifuge tube.Add 5 μ L RNaseA, 37 ℃ of constant temperature keep 30min.Use equal-volume phenol respectively: chloroform: primary isoamyl alcohol (25: 24: 1), extracting twice repeatedly, uses chloroform/primary isoamyl alcohol (24: 1) extracting more once.Under the room temperature condition, the centrifugal 10min of 10000g gets supernatant liquor and goes in another centrifuge tube.Add 2/3 volume primary isoamyl alcohol, 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 liquor, with 70% washing with alcohol precipitation once, pours out ethanol, dries precipitation.Add 50 μ L TE (pH8.0) dissolution precipitations, gained solution is sample DNA solution.
Embodiment 2LE Green dyestuff Mix preparation and using method
The prescription 1 of LE Green dyestuff Mix solution is as follows: Na
2HPO
40.02mol/L; NaH
2PO
40.02mol/L; NaCl 0.20mol/L; EDTA2Na 2.5mmol/L; PVP0.1%; TritonX-100 0.1%; Eva Green 3.0 μ mol/L; LC Green 2.0 μ mol/L; Glycerine 5%; Transfer pH to 7.3 with phosphoric acid at last; All the other are distilled water.
The prescription 2 of LE Green dyestuff Mix solution is as follows: Na
2HPO
40.01mol/L; NaH
2PO
40.01mol/L; NaCl 0.15mol/L; EDTA2Na 1.5mmol/L; PVP 0.05%; TritonX-100 0.05%; Eva Green 2.0 μ mol/L; LC Green 1.0 μ mol/L; Glycerine 3%; Transfer pH to 7.0 with phosphoric acid at last; All the other are distilled water.
The prescription 3 of LE Green dyestuff Mix solution is as follows: Na
2HPO
40.03mol/L; NaH
2PO
40.03mol/L; NaCl 0.25mol/L; EDTA2Na 3.5mmol/L; PVP0.2%; TritonX-100 0.15%; Eva Green 4.0 μ mol/L; LC Green 3.0 μ mol/L; Glycerine 8%; Transfer pH to 7.5 with phosphoric acid at last; All the other are distilled water.
It is the mixed Mix solution of LC Green and Eva Green dyestuff that LE Green concentrates dyestuff.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 detected result meeting relatively good after preparation in a few hours.The preparation of embodiment 3DNA standardized solution, blank liquid and sample DNA solution
The preparation of standardized solution.Calf thymus DNA is commonly used to do typical curve, and preparation concentration is the solution of 400ng/mL.In the LEGreen Mix working fluid that isopyknic calf thymus DNA storage liquid is joined, thorough mixing, reference liquid ultimate density 200ng/ml.
The preparation of blank liquid.The sample buffer that adds equal volume in LE Green Mix working fluid, thorough mixing.
The preparation of sample DNA solution.The sample that adds equal volume in LE Green Mix working fluid, thorough mixing.
1) sets up typical curve
With final concentration concentration is that the standard DNA solution of 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL forwards in the 100 μ l trace detection wares, and lucifuge incubated at room 2-5min does not introduce bubble in the attention mobility process.Click " MeasureFluorescence ", the sample net result can be presented on the function machine display screen, and Fig. 1 is calf thymus DNA concentration and fluorescent value canonical plotting.
2) the multi-functional luminosity meter of calibration Modulus
Count example with the multi-functional luminosity of Modulus, adopt and calibrate with the 150ng/ml standard DNA.
Annotate: the single-point calibration tolerance range can be higher, with a standard optimization system identical or close with test sample concentration, for example, as if test sample concentration at 100ng/ml, be with 50～150ng/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, click " Measure Fluorescence ", the sample net result can be presented on the function machine display screen, adopt prescription 1, prescription 2, the prescription 3 of LE Green dyestuff Mix solution, the concentration that records genomic dna is respectively 155ng/ml, 163ng/ml, 159ng/ml.
Preparation calf thymus DNA concentration is the solution of 300ng/ml.In the LE Green Mix working fluid that isopyknic calf thymus DNA storage liquid is joined, thorough mixing, reference liquid ultimate density 150ng/ml.The employing aforesaid method is measured, and finally records concentration (three mean value) and is 149.8ng/ml.
Embodiment 6 sensitivity determinations
Preparation calf thymus DNA concentration is the solution of 6.4ng/ml.It is the solution of 3.2ng/ml, 1.6ng/ml, 0.8ng/ml, 0.4ng/ml that gradient dilution becomes concentration, in the LE Green Mix working fluid that isopyknic calf thymus DNA storage liquid is joined, thorough mixing, final concentration are 3.2ng/ml, 1.6ng/ml, 0.8ng/ml, 0.4ng/ml, 0.2ng/ml.Fixed through the multi-functional luminosity instrumentation of Modulus, obtain Fig. 2 calf thymus double-strandednucleic acid concentration sensitivity graphic representation, sensitivity can reach 0.4ng/ml (be lower than 0.4ng/ml, reading approaches 0).
Claims (9)
1. LE Green dye solution Mix, it contains following component:
Na
2HPO
4 0.01～0.03mol/L
NaH
2PO
4 0.01～0.03mol/L
NaCl 0.15～0.25mol/L
EDTA·2Na 1.5～3.5mmol/L
PVP 0.05-0.20％
TritonX-100 0.05-0.15％
Eva?Green 2.0～4.0μmol/L
LC?Green 1.0～3.0μmol/L
Glycerine 3～8%
Transfer pH to 7.0～7.5 with phosphoric acid at last
All the other are distilled water.
2. LE Green dye solution Mix as claimed in claim 1, it contains following component:
Na
2HPO
4 0.02mol/L
NaH
2PO
4 0.02mol/L
NaCl 0.20mol/L
EDTA·2Na 2.5mmol/L
PVP 0.1％
TritonX-100 0.1％
Eva?Green 3.0μmol/L
LC?Green 2.0μmol/L
Glycerine 5%
Transfer pH to 7.3 with phosphoric acid at last
All the other are distilled water.
3. claim 1 or 2 application of described LE Green dye solution Mix in the double chain nucleotide detection by quantitative.
4. double chain nucleotide quantitative detection method, it comprises step: mix the lucifuge incubated at room with claim 1 or 2 described employing LE Green dyestuff Mix with double chain nucleotide solution to be measured, detect its fluorescence intensity, by comparing, obtain the concentration of double chain nucleotide solution to be measured with typical curve.
5. method as claimed in claim 4 is characterized in that, incubation time is 2～5min.
6. 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, mix with LE Green dye solution Mix respectively then and hatch.
7. as claim 4 or 5 described methods, it is characterized in that described typical curve is to be that standard substance is set up with the calf thymus DNA.
8. 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 fluorescent signal.
9. 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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200910241487 CN101724295B (en) | 2009-12-03 | 2009-12-03 | LE Green mixed dye and applications thereof |
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|---|---|---|---|
| CN 200910241487 CN101724295B (en) | 2009-12-03 | 2009-12-03 | LE Green mixed dye and applications thereof |
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|---|---|
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| CN101724295B CN101724295B (en) | 2013-03-13 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2565281A1 (en) * | 2011-08-29 | 2013-03-06 | Thermo Fisher Scientific Inc. | Dye blends |
| US20140302503A1 (en) * | 2013-03-08 | 2014-10-09 | Bio-Rad Laboratories, Inc. | Compositions, methods and systems for polymerase chain reaction assays |
| CN107884588A (en) * | 2017-11-13 | 2018-04-06 | 重庆艾维迪生物科技有限公司 | Hemolytic agent for detecting glycosylated hemoglobin and its preparation method and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008005246A2 (en) * | 2006-06-30 | 2008-01-10 | Canon U.S. Life Sciences, Inc. | Systems and methods for monitoring the amplification and dissociation behavior of dna molecules |
| GB0714058D0 (en) * | 2007-07-19 | 2007-08-29 | Abertec Ltd | detection of methylation in nucleic acid sequences |
| CN100554946C (en) * | 2007-12-18 | 2009-10-28 | 中国农业大学 | A kind of method of double stranded nucleic acid precision quantifying |
-
2009
- 2009-12-03 CN CN 200910241487 patent/CN101724295B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2565281A1 (en) * | 2011-08-29 | 2013-03-06 | Thermo Fisher Scientific Inc. | Dye blends |
| US20140302503A1 (en) * | 2013-03-08 | 2014-10-09 | Bio-Rad Laboratories, Inc. | Compositions, methods and systems for polymerase chain reaction assays |
| US9822393B2 (en) * | 2013-03-08 | 2017-11-21 | Bio-Rad Laboratories, Inc. | Compositions, methods and systems for polymerase chain reaction assays |
| US10676778B2 (en) | 2013-03-08 | 2020-06-09 | Bio-Rad Laboratories, Inc. | Compositions, methods and systems for polymerase chain reaction assays |
| CN107884588A (en) * | 2017-11-13 | 2018-04-06 | 重庆艾维迪生物科技有限公司 | Hemolytic agent for detecting glycosylated hemoglobin and its preparation method and application |
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|---|---|
| CN101724295B (en) | 2013-03-13 |
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