CN108037103B - Method for detecting DNA hybridization by using surface cationized R-phycoerythrin - Google Patents
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Abstract
The invention belongs to the technical field of nucleic acid detection, and discloses a method for detecting DNA hybridization by using surface cationized R-phycoerythrin. The method comprises the following specific steps: carrying out centrifugal ultrafiltration on R-phycoerythrin, desalting and diluting; then adding a cationic polymer PDADMAC to positively charge the surface of the protein; adding a single-stranded DNA modified by BHQ2 with a quenching group, wherein the fluorescence is quenched to about 88%; fluorescence is released continuously after adding different concentrations of the fully complementary strand. The invention has the advantages that: the natural plant protein R-PE and BHQ2 modified nucleic acid chain is used as donor and acceptor for fluorescence resonance energy transfer, cationic polymer is added to raise FRET efficiency, and when the nucleic acid chain is in free state, the fluorescence is quenched, and once the nucleic acid chain is combined with target chain, the fluorescence is recovered immediately, and its sensitivity is high, so that the goal of DNA hybridization detection can be reached.
Description
The technical field is as follows:
the invention belongs to the field of nucleic acid detection, and particularly relates to a method for detecting DNA hybridization by using surface cationized R-phycoerythrin.
Background art:
the R-phycoerythrin (R-phycoerythrin) is separated, purified and extracted from algae, is a novel fluorescent labeling material commonly used at present, has the fluorescence intensity 30-100 times of that of fluorescein, and has good light absorption performance and high quantum yield. The phycoerythrin is used for fluorescence analysis, and has incomparable superiority compared with the traditional chemical fluorescent dye: 1. the fluorescence emission material has a wide absorption spectrum in a wide PH range, and is easy to select a proper excitation wavelength, so that high-efficiency fluorescence emission is obtained, and a specific fluorescence emission peak is generated during excitation; 2. the absorbance and the fluorescence quantum yield are high, the fluorescence intensity is stable, and the sensitivity is high; 3. the fluorescent material has smaller fluorescent background, is not easy to quench, and has longer fluorescent retention period; 4. the water solubility is excellent, other molecules are easy to be cross-linked and combined, and the non-specific adsorption is less; 5. the pure natural marine organism extract has no toxic or side effect, no radioactivity and safe operation and use.
Pdadmac (poly dimethyl diallyl ammonium chloride) is a chemical substance. The product is strong cationic polyelectrolyte, and is colorless to light yellow viscous liquid. Safe, nontoxic, easily soluble in water, non-flammable, strong cohesive force, good hydrolytic stability, no gel formation, insensitivity to pH value change and chlorine resistance. The freezing point is about-2.8 ℃, the specific gravity is about 1.04g/cm, the decomposition temperature is 280-300 ℃, and the fluorescent property of phycoerythrin is not influenced by the temperature.
At present, research for developing DNA hybridization detection methods has become a hot spot at home and abroad, and most of them utilize nano materials for detection, such as gold gel, quantum dots, carbon nanotubes, upconversion nanoparticles, and the like. H Peng et al detect DNA hybridization using blue quantum dots and dye-labeled single-stranded DNA synthesis probes (Peng H, Zhang L,
T H,etal.DNAhybridization detection with blue luminescent quantum dots and dye-labeled single-stranded DNA[J]journal of the American Chemical Society,2007,129(11): 3048-9.). R Yang et al use carbon nanotube fluorescence quenching mononucleotide probes to achieve detection of DNA hybridization (Yang R, Jin J, Chen Y, et al, carbon nanotube-sequenced fluorescent oligonucleotides, probes which are fluorescent probes on upper hybridization]Journal of the American Chemical Society,2008,130(26): 8351.). HLi et al used the electrostatic interaction between unmodified gold nanoparticles and DNA sequences to detect DNA hybridization by colorimetry (Li H, Rothberg L. colorimetric detection of DNA sequences based on electrochemical hybridization nanoparticles [ J.].Proceedings of the National Academy ofSciences of the United States of America,2004,101(39):14036.)。
To date, no technique has been devised for detecting DNA hybridization using fluorescent proteins. Therefore, the present patent invented a new method for detecting DNA hybridization, i.e., using phycoerythrin whose surface is cationized to detect DNA hybridization.
The invention content is as follows:
the present invention aims to provide a novel method for detecting DNA hybridization.
In order to achieve the above object, the present invention provides a method for cationizing the surface of R-phycoerythrin and then detecting DNA hybridization. Firstly, relates to a method for cationizing the surface of R-phycoerythrin, which comprises the following steps:
(1) centrifuging, ultrafiltering and desalting R-phycoerythrin at 4 deg.C;
(2) diluting the concentration of phycoerythrin to 1mg/mL by using PBS buffer solution;
(3) mixing cationic polymer PDADMAC (weight)<100000，35wt.％in H2O) diluting with PBS buffer solution by 10 times;
(4) mixing R-PE and PDADMAC uniformly in a volume ratio of 1: 10.
The buffer used in steps (1) - (4) was 50mMPBS buffer, pH 7.5;
the centrifugation condition in the step (1) is preferably 3800rcf, and the centrifugation is carried out for 25-30 min;
the preferable ultrafiltration frequency in the step (1) is 4-5 times, and an ultrafiltration tube is 100 kD;
secondly, relates to a method for detecting DNA hybridization, which comprises the following steps:
(a) measuring the fluorescence intensity of the surface cationized R-PE at 575nm by using a fluorescence spectrometer;
(b) mixing and reacting the surface cationized R-PE with a DNA chain modified by a quenching group BHQ2 for 2 minutes at a ratio of 1:5, and measuring the fluorescence intensity of the DNA chain at 575 nm;
(c) uniformly mixing the DNA chain modified by BHQ2 and the complete complementary chain thereof in different proportions, then adding surface cationized R-PE, and measuring the fluorescence intensity of the DNA chain at 575nm after 2 minutes;
(d) the BHQ2 modified DNA single strand and the single base mismatched complementary strand are mixed uniformly in the ratio of 1:1, then the surface cationized R-PE is added, and the fluorescence intensity at 575nm is measured after 2 minutes.
The instrument for measuring the fluorescence intensity in the steps (a) - (d) is an Edinburgh integrated steady-state transient fluorescence spectrometer FS5, and the detection sensitivity is high;
the sequence of the DNA strand in step (b) is: 5'-TACGAGTTGAGAATCCTGAATGCG-BHQ 2-3', other sequences are also possible;
the sequence of the complementary strand of DNA in step (c) is: 5'-ATGCTCAACTCTTAGGACTTACGC-3', the sequence of which is transformed according to the sequence in step (b);
the sequence of the single base mismatching DNA complementary strand in the step (d) is as follows: 5'-ATGCTCAACTCGTAGGACTTACGC-3', the sequence of which is transformed according to the sequence in step (b)
Compared with the existing DNA hybridization detection technology, the invention has the advantages that:
(1) the R-phycoerythrin is a natural plant protein, has no toxicity, and is low in price and easy to obtain compared with other nano-particles;
(2) compared with other common methods, the method has the advantages of short sample preparation time and simple steps.
(3) High detection sensitivity and low detection limit.
Drawings
FIG. 1 is a schematic diagram of DNA hybridization detection.
FIG. 2 is a graph showing the effect of PDADMAC, a cationic polymer, on the fluorescence intensity of R-PE itself.
FIG. 3 is a graph showing the effect of a single-stranded DNA modified with a quenching group BHQ2 on fluorescence intensity.
FIG. 4 is a graph showing fluorescence release when different concentrations of the perfectly complementary strand were added.
FIG. 5 is a fluorescence image of the DNA with the addition of the perfectly complementary strand (a), the single-base mismatched strand (b), and the single-stranded DNA (c).
Detailed Description
In order to clearly understand the technical contents of the present invention, the following further describes a specific implementation method of the present invention.
The first embodiment is as follows: preparation of various solutions
(1) Preparation of 50mM PBS buffer
29.0g Na2HPO4-12H2O,2.9g NaH2PO4-2H2O,5.85g NaCl was dissolved in 900ml of ultrapure water, and the solution was diluted 2-fold in a 1000ml volumetric flask, to obtain a 50mM solution.
(2) Preparation of phycoerythrin solution
Transferring 100 μ L phycoerythrin crystal, centrifuging at 3800rcf 4 deg.C for 15min, removing supernatant, and dissolving precipitate with 500 μ L50mM sodium phosphate buffer (pH7.5) for 30 min; selecting an ultrafiltration tube with a 100kD filter membrane; the desalting was repeated 4 times at 3800rcf 4 ℃ by centrifugal ultrafiltration for 15min, after which the concentration was diluted to 1mg/mL with PBS buffer (pH 7.5).
(3) Preparation of cationic Polymer PDADMAC solution
PDADMAC (weight <100000, 35 wt.% in H2O) solution was diluted 10-fold with PBS buffer, at 50 mM.
(4) Preparation of solution of BHQ 2-modified DNA Single Strand
And centrifuging the synthesized DNA chain before uncovering the cover, wherein the rotating speed is 4000rpm, and the time is 30-60 s. Then, 44.5. mu.L of PBS buffer was added to each OD, and the stock solution was prepared to 100. mu.M and stored in a refrigerator at 4 ℃ and diluted to 1. mu.M at the time of use.
(5) Optimization of cationic polymer PDADMAC
Mu. L R-PE was mixed with 5. mu.L of DNA and reacted for 2 minutes, after which it was diluted to 100. mu.L with PBS buffer and allowed to stand for one minute, and then its fluorescence intensity at 575nm was measured with a fluorescence spectrometer. Then 1 μ L of PDADMAC solution is added into the system, the fluorescence intensity is measured, and then 1 μ L of PDADMAC solution is added each time until the fluorescence intensity at 575nm does not change any more, and the concentration of the PDADMAC solution at the time is the optimal value, namely 10 mM.
Example two: method for detecting DNA hybridization
(1) After R-PE and PDADMAC solution in a volume ratio of 1:10 are mixed uniformly, 5 mu L of single-stranded DNA solution is added, then the mixture is diluted to 100 mu L by PBS buffer solution, and the fluorescence intensity at 575nm is measured after two minutes, at which time the fluorescence intensity is observed to be quenched by about 88%.
(2) 5 mu L of single-stranded DNA solution and completely complementary strand solution with different concentrations are mixed evenly, 1 mu L of R-PE and 10 mu L of PDADMAC solution are added, the mixture is diluted to 100 mu L after reacting for 2 minutes, the fluorescence intensity of the mixture at 575nm is measured after two minutes, and the gradual release of the fluorescence intensity can be observed at the moment, and the fluorescence intensity is basically the same as that of the protein.
(3) mu.L of single-stranded DNA solution and 5 mu.L of single-base mismatched complementary strand solution are mixed uniformly, 1 mu.L of R-PE and 10 mu.L of PDADMAC solution are added, the mixture is diluted to 100 mu.L after reacting for 2 minutes, the fluorescence intensity of the mixture at 575nm is measured after two minutes, and the fluorescence intensity is observed to be quenched by about 20 percent.
Therefore, the detailed method can distinguish single double-stranded and single base mismatching, thereby achieving the purpose of DNA hybridization detection.
Claims (6)
1. A method for detecting DNA hybridization by using surface cationized R-phycoerythrin is characterized in that:
(1) centrifuging, ultrafiltering and desalting R-phycoerythrin at 4 deg.C;
(2) uniformly mixing R-phycoerythrin and a cationic polymer PDADMAC according to the molar concentration of 1: 50;
(3) respectively detecting the fluorescence intensity of the R-phycoerythrin before and after cationization by using an Edinburgh integrated steady-state transient fluorescence spectrometer FS 5;
(4) uniformly mixing the mixed solution in the step (2) with a DNA single-chain solution modified by a quenching group BHQ2 according to the molar concentration ratio of 1:5, diluting to 100 mu L, standing for 2 minutes, measuring the fluorescence intensity, and observing that the fluorescence is quenched by about 88%;
(5) uniformly mixing a DNA single-chain solution modified by a quenching group BHQ2 and an unmodified complete complementary chain solution in a molar concentration ratio of 1:1, uniformly mixing the solution in the step (2) and the solution in a molar concentration ratio of 1:5, diluting to 100 mu L, standing for 2 minutes, measuring the fluorescence intensity, and observing that the fluorescence intensity is not changed basically;
(6) uniformly mixing a DNA single-chain solution modified by a quenching group BHQ2 and an unmodified complete complementary chain solution in a molar concentration ratio of 5:1, 5:2, 5:3, 5:4 and 1:1 respectively, then uniformly mixing the solution of (2) and the solution of (2) in a molar concentration ratio of 1:5, diluting to 100 mu L, standing for 2 minutes, measuring the fluorescence intensity, and observing that the fluorescence intensity is gradually released and is finally basically the same as that of R-phycoerythrin;
(7) and (3) uniformly mixing a DNA single-chain solution modified by a quenching group BHQ2 and an unmodified single-base mismatched complementary chain solution in a molar concentration ratio of 1:1, uniformly mixing the solution in the step (2) and the solution in a molar concentration ratio of 1:5, diluting to 100 mu L, standing for 2 minutes, measuring the fluorescence intensity, and observing that the fluorescence intensity is quenched by about 20%.
2. The method of claim 1, wherein the method comprises the following steps: the buffer used for dilution in steps (1) to (7) was 50 mpbs buffer at pH 7.5.
3. The method of claim 1, wherein the method comprises the following steps: the centrifugation condition in the step (1) is preferably 3800rcf, and the centrifugation is carried out for 25-30 min.
4. The method of claim 1, wherein the method comprises the following steps: the preferable ultrafiltration frequency in the step (1) is 4-5 times, and the size of an ultrafiltration tube is 100 kD.
5. The method of claim 1, wherein the method comprises the following steps: the fluorescence intensity detection in the steps (3) to (7) is carried out at an excitation wavelength of 535nm and an emission wavelength of 575 nm.
6. The method of claim 1, wherein the method comprises the following steps: the single-stranded DNA modified by BHQ2 in the steps (4) to (7) may be a single-stranded DNA having any sequence.
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