CN100590202C - Method for realizing single base mispairing identification using nano gold particles and electroelution - Google Patents
Method for realizing single base mispairing identification using nano gold particles and electroelution Download PDFInfo
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- CN100590202C CN100590202C CN200610032471A CN200610032471A CN100590202C CN 100590202 C CN100590202 C CN 100590202C CN 200610032471 A CN200610032471 A CN 200610032471A CN 200610032471 A CN200610032471 A CN 200610032471A CN 100590202 C CN100590202 C CN 100590202C
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Abstract
The nanometer gold particle and electroelution process for identifying single base mispairing includes the following steps: fixing DNA probe, adding target DNA, flushing repeatedly, and detecting theSPR responding signal of target DNA hybridization; adding sulfhydryl DNA modified nanometer gold particle hybridization, flushing repeatedly, and detecting the SPR responding signal after sulfhydryl DNA hybridization; negatively charging the metal film surface for electroelution and detecting the responding signal after electroelution; and judging whether to have single base mispairing DNA of theadded target DNA based on whether the SPR responding signal after electroelution is smaller than the SPR responding signal after sulfhydryl DNA hybridization. The process of the present invention caneliminate the interference of the single base mispairing target DNA and ensure no influence on the cDNA hybridizing signal, and has simple DNA probe design and reuseable sensor chip.
Description
Technical field
The invention belongs to a kind of method of nucleic acid mismatch identification, be specifically related to a kind of method that realizes single base mismatch identification.
Background technology
Single nucleotide polymorphism (SNP) is meant the dna sequence polymorphism that single nucleotide diversity causes on the genomic level.The polymorphism that SNP showed only relates to the sudden change of single base, and this sudden change can be caused that by the conversion or the transversion of single base the ratio of the two is about 2: 1.Single base mutation can cause the difference between colony and colony, individuality and the individuality.The drug susceptibilities of the susceptibility of numerous disease, individuality etc. are all relevant with single base mutation.The research single base mutation helps to explain individual performance difference, different groups and individual to disease, particularly to the susceptibility of complex disease, and to the tolerance of various medicines with to the reaction of environmental factor, for the exploitation of working out proper treatment scheme, medicine provides foundation.
The nucleic acid sensing technology has become the important means of studying nucleic acid owing to have characteristics such as quick, sensitive, simple to operate, pollution-free, fields such as the development of be widely used in suddenling change detection, genescreen, gene diagnosis, medicine and exploitation.But in the nucleic acid sensing technology,, cause that signal changes, thereby disturb the detection of complete complementary DNA (cDNA) and false positive results occurs because the DNA of single base mismatch can carry out part hybridization with the immobilized DNA probe.Usually adopt the rigorous degree (as temperature, ionic strength, methane amide concentration etc.) of regulating hybridization conditions, carry out wash-out in the rigorous degree condition of height and distinguish single base mismatch DNA, change but this method might reduce the signal that cDNA hybridization causes when reducing the influence that single base mismatch DNA causes.In addition, adopt the good nucleic acid probe of some specificitys (as peptide nucleic acid probe, molecular beacon) to replace the ssDNA probe also can improve the ability of identification form base mispairing, but this method exist probe design and preparation relative complex, the high deficiency of cost.
Summary of the invention
Technical problem to be solved by this invention is the defective that overcomes above-mentioned prior art, provides that a kind of simple and easy to do, detected result is stable, the nucleic acid probe simplicity of design, does not reduce that the signal that cDNA hybridization causes changes, made sensing chip can reuse, cost is low and can utilize nm gold particles fast and accurately and electroelution is realized the method for single base mismatch identification.
For solving the problems of the technologies described above, the inventive method with surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) transmitter is as detection means and adopt following technical proposals:
A. fixing DNA probe on surface plasma resonance sensor metallic film surface, make sensing chip, adding target dna then hybridizes, neutral buffered solution washes metallic film repeatedly, utilizes the surface plasma resonance response signal after surface plasma resonance sensor obtains target dna hybridization;
B. add the nm gold particles of sulfydryl dna modification in the above-mentioned final gained system, after hybridization was finished, neutral buffered solution washed metallic film repeatedly, utilized the surface plasma resonance response signal after surface plasma resonance sensor obtains sulfydryl DNA hybridization;
C. regulate the current potential official post metallic film surface bear between metallic film and the conductive glass, wash metallic film repeatedly with neutral buffered solution then and carry out electroelution, utilize the surface plasma resonance response signal after surface plasma resonance sensor obtains electroelution;
D. surface plasma resonance response signal after surface plasma resonance response signal, the sulfydryl DNA after the comparison object DNA hybridization hybridized and the surface plasma resonance response signal behind the electroelution, the surface plasma resonance response signal after if the surface plasma resonance response signal behind the electroelution is hybridized less than sulfydryl DNA, then the target dna that adds as can be known is the DNA of single base mismatch.
Utilize the method for covalent cross-linking dna probe to be fixed in the metallic film surface of spr sensor among the described step a; The method of described covalent cross-linking can be amino coupled method, sulfydryl coupling method or biotin-avidin cross-linking method; Described dna probe can be amino dna probe, sulfydryl dna probe or biotinylated dna probe; 6 * SSC that described neutral buffered solution is pH 7.0 (6 times of Saline-Sodium Citrate) buffered soln; The nm gold particles particle diameter of described sulfydryl dna modification is 10～20nm; Described metallic film is golden film; Described conductive glass is tin indium oxide (ITO) conductive glass; Among the described step c, the current potential of the relative conductive glass of metallic film is-5～-20V, the electroelution time is 5～15 minutes.
If the target dna that adds is the DNA of single base mismatch, behind the electroelution, the dsDNA that the nm gold particles hybridization of the DNA of single base mismatch and sulfydryl dna modification forms is untwisted, the nm gold particles of sulfydryl dna modification is cushioned solution and rinses out, thereby cause the variation of system SPR response signal, the SPR response signal behind the electroelution is less than the SPR response signal after the sulfydryl DNA hybridization; If the target dna that adds is cDNA, behind the electroelution, the dsDNA that forms with the hybridization of the nm gold particles of sulfydryl dna modification can not untwist, thereby can not cause the variation of system SPR response signal yet, and the SPR response signal behind this moment electroelution is identical with the SPR response signal after the sulfydryl DNA hybridization.Therefore, can obviously distinguish single base mismatch DNA and cDNA, thereby realize the identification of single base mismatch DNA according to the variation of system SPR response signal.
The advantage of the inventive method is: both can remove the interference that the single base mismatch target dna is caused by electroelution 1. simple and effectively, it is unaffected in electroelution to guarantee that again signal that cDNA hybridization causes changes, and distinguishes single base mismatch target dna and cDNA easily; 2. the dna probe simplicity of design that adopts in the inventive method, synthetic convenient, preparation cost is low; Used sensing chip can reuse, and has reduced cost; 3. the inventive method is simple and easy to do, cost is low, not only can adopt in spr sensor, can also expand to the other biological transmitter; 4. the inventive method has been not only for identification form base mispairing DNA provides a kind of new means, and provides a kind of new thinking for the biosensor that makes up highly selective.
Description of drawings
Fig. 1: nm gold particles and electroelution effect combine to detect and mate the target dna principle schematic fully.
Fig. 2: nm gold particles and electroelution effect combine and detect single base mismatch target dna principle schematic.
Fig. 3: nm gold particles and electroelution effect combine and detect the SPR spectrogram that mates target dna fully.
Fig. 4: nm gold particles and electroelution effect combine and detect the SPR spectrogram of single base mismatch target dna.
Fig. 5: the influence of the electroelution effect dsDNA that hybridization forms to the single base mismatch target dna.
More than each number in the figure represent:
1, golden film 2, dna probe 3, mate target dna fully
4, nm gold particles 41, the marking sulfhydryl dna probe of the modification of sulfydryl dna probe
42, nm gold particles 5, under the bear electricity condition of golden film surface, carry out electroelution
6, single base mismatch target dna
A, amido modified dna probe are fixed on the lip-deep SPR spectrum of golden film
B, mate SPR spectrum after the target dna hybridization fully with 33nM
B ', with 33nM single base mismatch target dna
1SPR spectrum after the hybridization
SPR spectrum after c, the nm gold particles reaction of modifying with the marking sulfhydryl dna probe
SPR spectrum behind d, the electroelution
SPR spectrum after e, the manipulation of regeneration
A, mate target dna B, single base mismatch target dna fully
1
C, single base mismatch target dna
2D, single base mismatch target dna
3
E, single base mismatch target dna
4F, single base mismatch target dna
5
Resonant wavelength shift value (λ before I, the electroelution
2-λ
1)
Resonant wavelength shift value (λ after II, the electroelution
3-λ
1)
Embodiment
(this sensor arrangement is as follows: comprise light source, collimator tube, p polaroid, prism, prism bracket, flow-through cell, adjustable direct supply, grating spectrograph and computer based on the surface plasma resonance sensor of wavelength-modulated in employing, prism by screw retention in prism bracket, light source places before the collimator tube, the p polaroid is bonded at the exit portal of collimator tube, place the prism of prism bracket to be positioned at after the collimator tube, grating spectrograph is arranged on the prismatical emitting light path, and links to each other with the interface of computer by data line.Light source, collimator tube and prism bracket are fixed on the base by adjustable platform respectively, and grating spectrograph and computer are placed on the base.Flow-through cell places the prism top, this flow-through cell is made up of golden film and conductive glass, the gold film is bonded on the prism bottom surface by index-matching oil such as cedar oil, prism is the isosceles right angle prism in the present embodiment, also can be the isosceles trapezoid prism, golden film is bonded in prismatical bottom surface (being face relative with the right angle in these isosceles right angle prism).The glass coating bottom surface and the golden film of conductive glass are bonding, the glass coating bottom of conductive glass offers the sample introduction groove, the degree of depth of sample introduction groove is 0.05～0.15mm, length equals the length of conductive glass, form sample intake passage between glass coating and golden film, the conductive layer that is positioned at the conductive glass of glass coating end face links to each other with adjustable direct supply by lead respectively with golden film.Light source adopts halogen tungsten lamp in this example; Grating spectrograph is made up of plane grating, quasi-optical system, charge-coupled device (CCD) detector, and the spectral response interval of charge-coupled device (CCD) detector is 300～900nm.
The working process of this transmitter is: the light path of adjusting this device by adjustable platform, the light beam process collimator tube that light source is sent, inject the isosceles right angle prism behind the p polaroid, be coupled with the golden film on the prism, reflected light incides grating spectrograph, quasi-optical system in grating spectrograph incides on the plane grating of grating spectrograph, plane grating projects the collimated monochromatic ligth that light beam is divided into different wave length on the pale pinkish purple device of electric charge (CCD) detector of grating spectrograph, the pale pinkish purple device of electric charge (CCD) detector is converted to electrical signal with optical signal, is transferred to by data line after mould/number conversion and obtains corresponding spectrogram in the computer.) as detection means, it is the displacement of resonant wavelength (λ) that response signal changes.
Embodiment one:
1. golden film surface needs to clean before modification, cleans up with acetone, dehydrated alcohol and secondary water at first successively, and be the vitriol oil/30% hydrogen peroxide solution cleaning 1-2min of 3: 1 then with volume ratio, rinse well repeatedly with secondary water again, use N
2Dry up, promptly can be used for finishing.The step of the modification of gold film is as follows: (1) at first handles 70min at least with 10mmol/L thiohydracrylic acid/ethanol solution, uses 0.01mol/L phosphoric acid buffer (pH=7.3) to wash the thiohydracrylic acid that flush away does not adsorb repeatedly again; (2) use 100mmol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxy-succinamide at room temperature reaction 60min then, then use 0.01mol/L phosphoric acid buffer (pH=7.3) damping fluid to rinse well repeatedly; (3) injecting the amido modified dna probe 2 of 0.57 μ mol/L (buying in precious biotechnology Dalian company limited) at last at room temperature cultivates, wash repeatedly with 0.01mol/L phosphoric acid buffer (pH=7.3) after for some time, amido modified dna probe 2 can be fixed in golden film 1 surface of surface plasma resonance sensor, obtain the DNA sensing chip.Amido modified dna probe sequence is 5 '-CGCCTCACAACCAAAAAA-C
6H
12-NH
2-3 '.Record SPR spectrum at this moment is curve a, its resonant wavelength λ
0Be 663.0nm (as shown in Figure 3 and Figure 4);
2. as shown in figures 1 and 3, adding 33nM on step 1 basis mates target dna (cDNA) 3 fully and hybridizes, hybridization time is 30min, the sequence of cDNA is 5 '-GGTTGTGAGGCGCTGCCCAAGCGA-3 ', afterwards, (this 6 * SSC is made up of 0.9mol/L sodium chloride and 0.09mol/L Trisodium Citrate with 6 * SSC of pH 7.0, and the pH that regulates this solution for 0.1mol/L hydrochloric acid with concentration is 7.0) after buffered soln washes repeatedly, find that SPR spectrum moves towards the long wave direction from curve a, move to curve b, the resonant wavelength λ of curve b
1Be 665.4nm, then resonant wavelength displacement (λ
1-λ
0) be 2.4nm, illustrate that cDNA has been trapped in golden film surface; The particle diameter that then adds excessive sulfydryl dna modification is the nm gold particles 4 of 13nm (the nm gold particles preparation method of sulfydryl dna modification: at first prepare nm gold particles with trisodium citrate reduction aqueous solution of chloraurate method, concrete steps are as follows: accurately pipette 100mL 0.01% chlorauric acid solution in beaker, be heated to and boil, under vigorous stirring, add 1% citric acid three sodium solution that 3mL newly joins fast, boil about 5min continuously, solution colour is by the dark blue redness that becomes, and keep stable, promptly obtain red nano-Au solution.With being diluted to the 100mL volumetric flask after the nano-Au solution room temperature cooling for preparing, shake up, place 4 ℃ of environment to preserve.Then at nm gold particles finishing sulfydryl DNA, step is as follows: at first get 100 μ L, 36 μ mol/L sulfydryl DNA and mix with the above-mentioned nano-Au solution of 900 μ L, under 4 ℃ of conditions, cultivate 16h, add sodium-chlor and phosphate buffer solution (pH 7.0) (formulated) then by potassium primary phosphate and two kinds of salt of Sodium phosphate dibasic, make mixture aging 40h under 4 ℃ of conditions of above-mentioned nm gold particles-sulfydryl DNA, the final concentration of sodium-chlor and phosphate buffer solution (pH 7.0) is respectively 0.1mol/L and 0.01mol/L.Then with after the centrifugal 30min of the speed of 13000rpm, abandoning supernatant, red oily precipitation at the bottom of the centrifuge tube is loose in the 0.01mol/L phosphate buffer solution that contains 0.1mol/L sodium-chlor (pH 7.0), again with the centrifugal 30min of the speed of 13000rpm, repeat twice washing, unconjugated sulfydryl DNA is removed.The nm gold particles of the sulfydryl dna modification that has washed is dispersed in the 0.01mol/L phosphate buffer solution (pH 7.0) that contains 0.3mol/L sodium-chlor, and it is standby to place 4 ℃ of environment to preserve.Wherein, the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 '.), reaction 30min after 6 * SSC (pH 7.0) buffered soln washes repeatedly, finds that SPR spectrum continues to move towards the long wave direction, moves to curve c, the resonant wavelength λ of curve c
2Be 672.1nm, resonant wavelength displacement (λ
2-λ
1) be 6.7nm, reflection nm gold particles this moment has been fixed on golden film surface.Connect circuit then, the current potential of regulating golden film (relatively ITO conductive glass) is-10V, and washes 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, writing down SPR spectrum is curve d, curve d is identical with the resonant wavelength of curve c.This illustrates that utilizing electroelution can not influence substantially by the sulfydryl DNA on cDNA and nm gold particles surface hybridizes the dsDNA that forms under this strength of electric field.Utilize 90 ℃ of hot water to clean 5min at last, use after 6 * SSC (pH 7.0) buffered soln washes repeatedly again, find that SPR spectrum moves towards the shortwave direction, move to curve e, curve e is identical with the resonant wavelength of curve a, and the hot water sensing chip of can regenerating is described.
3. as Fig. 2 and shown in Figure 4, on step 1 basis, add 33nM single base mismatch target dna
16 hybridize, and hybridization time is 30min, the single base mismatch target dna
1Sequence be 5 '-GGTTGTGAGGCG
GTGCCCAAGCGA-3 ' afterwards, washes repeatedly with 6 * SSC (pH 7.0) buffered soln, and discovery can cause that SPR spectrum moves towards the long wave direction from curve a, moves to curve b ', the resonant wavelength λ of curve b '
1Be 665.3nm, then resonant wavelength displacement (λ
1-λ
0) be 2.3nm, instruction book base mispairing target dna
1Be trapped in golden film surface; The particle diameter that then adds excessive sulfydryl dna modification is the nm gold particles of 13nm, reacts 30min, after washing repeatedly with 6 * SSC (pH7.0) buffered soln, finds that SPR spectrum continues to move towards the long wave direction, moves to curve c, the resonant wavelength λ of curve c
2Be 669.5nm, resonant wavelength displacement (λ
2-λ
1) be 4.2nm, reflection nm gold particles this moment also is trapped in golden film surface.Wherein, the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 '.Connect circuit then, the current potential of regulating golden film (ITO conductive glass relatively) is-10V, and wash 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, record SPR spectrum is curve d, can find that SPR spectrum obviously moves towards the shortwave direction, the resonant wavelength of curve d and resonant wavelength less than curve c.This explanation utilizes electroelution can make by single base mismatch DNA under this strength of electric field
1Hybridizing the dsDNA that forms with the sulfydryl DNA on nm gold particles surface untwists, the nm gold particles of the sulfydryl dna modification that is untwisted is rinsed with buffered soln, cause that SPR spectrum obviously moves towards the shortwave direction, get back to the nm gold particles state before that does not add the sulfydryl dna modification.Clean 5min with 90 ℃ of hot water at last, use after 6 * SSC (pH 7.0) buffered soln washes repeatedly again, find that SPR spectrum moves towards the shortwave direction, move to curve e, curve e is identical with the resonant wavelength of curve a, and the hot water sensing chip of can regenerating is described.
Hence one can see that, when the current potential of golden film (for the ITO conductive glass) be-during 10V, utilizing electroelution that the sulfydryl DNA on cDNA and nm gold particles surface is hybridized the resonant wavelength displacement that causes does not have influence (resonant wavelength of the resonant wavelength of curve d and curve b in the comparison diagram 3), and to single base mismatch DNA
1, under this strength of electric field, utilize electroelution can remove by single base mismatch DNA
1Hybridize the resonant wavelength displacement (resonant wavelength of the resonant wavelength of curve d and curve b ' in the comparison diagram 4) that causes with the sulfydryl DNA on nm gold particles surface.Therefore, can utilize nm gold particles and electroelution to realize the single base mismatch target dna
1Identification.
Embodiment two:
1. utilize the golden film surface of being fixed in surface plasma body resonant vibration (SPR) transmitter as the dna probe 2 that 0.57 μ mol/L is amido modified of step 1 method among the embodiment one, the sequence of amido modified dna probe is 5 '-CGCCTCACAACCAAAAAA-C
6H
12-NH
2-3 ', writing down its SPR spectrum is curve 1, and its resonant wavelength is λ
0
2. add 33nmol/L single base mismatch target dna then
2Hybridize, hybridization time is 30min, the single base mismatch target dna
2Sequence be 5 '-GGTTGTGAGGCG
ATGCCCAAGCGA-3 ' afterwards, washes repeatedly with 6 * SSC (pH 7.0) buffered soln, and discovery can cause that SPR spectrum moves towards the long wave direction from curve 1, moves to curve 2, the resonant wavelength λ of curve 2
1The particle diameter that then adds excessive sulfydryl dna modification is the nm gold particles of 13nm, reaction 30min, and the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 ' washes golden film repeatedly with 6 * SSC (pH 7.0) buffered soln, finds that SPR spectrum continues to move towards the long wave direction, moves to curve 3, and the resonant wavelength of curve 3 is λ
2, resonant wavelength displacement (λ
2-λ
1) be 4.4nm (seeing the I part of C among Fig. 5).
3. connect circuit then, the current potential of regulating golden film (ITO conductive glass relatively) is-8V, and washes 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, record SPR spectrum is curve 4, curve 4 obviously moves towards the shortwave direction, the resonant wavelength λ of curve 4
3Resonant wavelength λ less than curve 3
2, resonant wavelength displacement (λ
3-λ
2) be-4.3nm resonant wavelength displacement (λ
3-λ
1) be 0.1nm (seeing the II part of C among Fig. 5).
This explanation utilizes electroelution to remove by single base mismatch DNA under this strength of electric field
2Hybridize the resonant wavelength displacement that causes with the sulfydryl DNA on nm gold particles surface, and for cDNA, utilize electroelution that the sulfydryl DNA on cDNA and nm gold particles surface is hybridized the resonant wavelength displacement that causes under this strength of electric field and do not have influence (I of A and II part in the comparison diagram 5), therefore, can utilize nm gold particles and electroelution to realize the single base mismatch target dna
2Identification.
Embodiment three:
1. with step 1 among the embodiment two.
2. add 33nmol/L single base mismatch target dna then
3Hybridize, hybridization time is 30min, the single base mismatch target dna
3Sequence be 5 '-GGTTGTGAGGCG
TTGCCCAAGCGA-3 ', after washing repeatedly with 6 * SSC (pH7.0) buffered soln, discovery can cause that SPR spectrum moves towards the long wave direction from curve 1, moves to curve 2, the resonant wavelength of curve 2 is λ
1The particle diameter that then adds excessive sulfydryl dna modification is the nm gold particles of 13nm, reaction 30min, and the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 ' uses after 6 * SSC (pH 7.0) buffered soln washes repeatedly afterwards, finds that SPR spectrum continues to move towards the long wave direction, moves to curve 3, and the resonant wavelength of curve 3 is λ
2, resonant wavelength displacement (λ
2-λ
1) be 4.3nm (seeing the I part of D among Fig. 5).
3) connect circuit then, the current potential of regulating golden film (ITO conductive glass relatively) is-15V, and washes 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, record SPR spectrum is curve 4, curve 4 obviously moves towards the shortwave direction, the resonant wavelength λ of curve 4
3Resonant wavelength λ less than curve 3
2, resonant wavelength displacement (λ
3-λ
2) be-4.1nm resonant wavelength displacement (λ
3-λ
1) be 0.2nm (seeing the II part of D among Fig. 5).This explanation utilizes electroelution can remove by the single base mismatch target dna under this strength of electric field
3Hybridize the resonant wavelength displacement that causes with the sulfydryl DNA on nm gold particles surface.And for cDNA, utilize electroelution that the sulfydryl DNA on cDNA and nm gold particles surface is hybridized the resonant wavelength displacement that causes under this strength of electric field and do not have influence (I of A and II part in the comparison diagram 5), therefore, can utilize nm gold particles and electroelution to realize the single base mismatch target dna
3Identification.
Embodiment four:
1. with step 1 among the embodiment two.
2. add 33nmol/L single base mismatch target dna then
4Hybridize, hybridization time is 30min, the single base mismatch target dna
4Sequence be 5 '-GGTTGTGAGGCG CTGCC
GAAGCGA-3 ' afterwards, washes repeatedly with 6 * SSC (pH 7.0) buffered soln, and SPR spectrum moves towards the long wave direction from curve 1, moves to curve 2, and the resonant wavelength of curve 2 is λ
1The particle diameter that then adds excessive sulfydryl dna modification is the nm gold particles of 13nm, reaction 30min, and the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 ', after washing repeatedly with 6 * SSC (pH7.0) buffered soln, SPR spectrum continues to move towards the long wave direction, moves to curve 3, and the resonant wavelength of curve 3 is λ
2, resonant wavelength displacement (λ
2-λ
1) be 1.3nm (seeing the I part of E among Fig. 5).
3. connect circuit then, the current potential of regulating golden film (ITO conductive glass relatively) is-5V, and washes 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, record SPR spectrum is curve 4, curve 4 obviously moves towards the shortwave direction, the resonant wavelength λ of curve 4
3Resonant wavelength λ less than curve 3
2, resonant wavelength displacement (λ
3-λ
2) be-1.2nm resonant wavelength displacement (λ
3-λ
1) be 0.1nm (seeing the II part of E among Fig. 5).This explanation utilizes electroelution can remove by the single base mismatch target dna under this strength of electric field
4Hybridize the resonant wavelength displacement that causes with the sulfydryl DNA on nm gold particles surface.And for cDNA, utilize electroelution that the sulfydryl DNA on cDNA and nm gold particles surface is hybridized the resonant wavelength displacement that causes under this strength of electric field and do not have influence (I of A and II part in the comparison diagram 5), therefore, can utilize nm gold particles and electroelution to realize the single base mismatch target dna
4Identification.
Embodiment five:
1. with step 1 among the embodiment two.
2. add 33nmol/L single base mismatch target dna then
5Hybridize, hybridization time is 30min, the single base mismatch target dna
5Sequence be 5 '-GGTTGTGAGGCG CTGCCCAAGCG
T-3 ', afterwards, to wash repeatedly with 6 * SSC (pH 7.0) buffered soln, discovery can cause that SPR spectrum moves towards the long wave direction from curve 1, moves to curve 2, the resonant wavelength of curve 2 is λ
1The particle diameter that then adds excessive sulfydryl dna modification is that the nm gold particles of 13nm reacts 30min, and the sequence of sulfydryl DNA is 5 '-HS-C
6H
12-AAAAAATCGCTTGGGCAG-3 ' washes repeatedly with 6 * SSC (pH 7.0) buffered soln, finds that SPR spectrum continues to move towards the long wave direction, moves to curve 3, and the resonant wavelength of curve 3 is λ
2, resonant wavelength displacement (λ
2-λ
1) be 3.9nm (seeing the I part of F among Fig. 5).
3. connect circuit then, the current potential of regulating golden film (ITO conductive glass relatively) is-8V, and washes 5min repeatedly with 6xSSC (pH 7.0) buffered soln, then, breaking circuits, record SPR spectrum is curve 4, curve 4 obviously moves towards the shortwave direction, the resonant wavelength λ of curve 4
3Resonant wavelength λ less than curve 3
2, resonant wavelength displacement (λ
3-λ
2) be-3.8nm resonant wavelength displacement (λ
3-λ
1) be 0.1nm (seeing the II part of F among Fig. 5).This explanation utilizes electroelution to remove by the single base mismatch target dna under this strength of electric field
5Hybridize the resonant wavelength displacement that causes with the sulfydryl DNA on nm gold particles surface.And for cDNA, utilize electroelution that the sulfydryl DNA on cDNA and nm gold particles surface is hybridized the resonant wavelength displacement that causes under this strength of electric field and do not have influence (I of A and II part in the comparison diagram 5), therefore, can utilize nm gold particles and electroelution to realize the single base mismatch target dna
5Identification.
Claims (7)
1, a kind of method of utilizing the single base mismatch identification that nm gold particles and electroelution realize non-diagnostic purpose is characterized in that may further comprise the steps:
A. fixing DNA probe on surface plasma resonance sensor metallic film surface, make sensing chip, adding target dna then hybridizes, neutral buffered solution washes metallic film repeatedly, utilizes the surface plasma resonance response signal after surface plasma resonance sensor obtains target dna hybridization;
B. add the nm gold particles of sulfydryl dna modification in the above-mentioned final gained system, after hybridization was finished, neutral buffered solution washed metallic film repeatedly, utilized the surface plasma resonance response signal after surface plasma resonance sensor obtains sulfydryl DNA hybridization;
C. regulate potential difference between metallic film and the conductive glass to-5～-20V, make metallic film surface bear, wash metallic film repeatedly with neutral buffered solution then and carry out electroelution, utilize the surface plasma resonance response signal after surface plasma resonance sensor obtains electroelution;
D. surface plasma resonance response signal after surface plasma resonance response signal, the sulfydryl DNA after the comparison object DNA hybridization hybridized and the surface plasma resonance response signal behind the electroelution, the surface plasma resonance response signal after if the surface plasma resonance response signal behind the electroelution is hybridized less than sulfydryl DNA, then the target dna that adds as can be known is the DNA of single base mismatch.
2, the method for discerning according to the described single base mismatch that utilizes nm gold particles and electroelution to realize non-diagnostic purpose of claim 1, it is characterized in that, utilize the method for covalent cross-linking dna probe to be fixed in the metallic film surface of surface plasma resonance sensor among the described step a.
3, the method for discerning according to the described single base mismatch that utilizes nm gold particles and electroelution to realize non-diagnostic purpose of claim 2 is characterized in that the method for covalent cross-linking is amino coupled method, sulfydryl coupling method or biotin-avidin cross-linking method; Described dna probe is amido modified dna probe, sulfydryl dna probe or biotinylated dna probe.
4, according to claim 1 or the 2 or 3 described methods of utilizing nm gold particles and electroelution to realize the single base mismatch of non-diagnostic purpose, it is characterized in that 6 * SSC buffered soln that described neutral buffered solution is pH 7.0.
5, according to claim 1 or the 2 or 3 described methods of utilizing the single base mismatches identification that nm gold particles and electroelution realize non-diagnostic purpose, it is characterized in that the nm gold particles particle diameter of described sulfydryl dna modification is 10～20nm.
6, according to claim 1 or the 2 or 3 described methods of utilizing the single base mismatches identification that nm gold particles and electroelution realize non-diagnostic purpose, it is characterized in that described metallic film is golden film.
7, according to claim 1 or the 2 or 3 described methods of utilizing the single base mismatches identification that nm gold particles and electroelution realize non-diagnostic purpose, it is characterized in that described conductive glass is an indium tin oxide-coated glass.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610032471A CN100590202C (en) | 2006-10-27 | 2006-10-27 | Method for realizing single base mispairing identification using nano gold particles and electroelution |
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| "Point mutation detection with the sandwichmethodemploying hydrogel nanospheres by the surfaceplasmonresonance imaging technique". Aya Okumura et al..Analytical Biochemistry,Vol.339 No.2. 2005 |
| "Point mutation detection with the sandwichmethodemploying hydrogel nanospheres by the surfaceplasmonresonance imaging technique". Aya Okumura et al..Analytical Biochemistry,Vol.339 No.2. 2005 * |
| 基于表面等离子体共振原理的核酸检测技术. 刘学勇等.航天医学与医学工程，第20卷第1期. 2007 |
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| 纳米金标记法在DNA检测及基因芯片技术重点应用. 谷宇，何农跃.中国药科大学学报，第34卷第2期. 2003 |
| 纳米金标记法在DNA检测及基因芯片技术重点应用. 谷宇，何农跃.中国药科大学学报，第34卷第2期. 2003 * |
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