CN103614459A - Non-labeled SNP detection method - Google Patents
Non-labeled SNP detection method Download PDFInfo
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Abstract
A disclosed non-labeled SNP detection method comprises: (1) providing two or more oligonucleotide probes, connecting at least one oligonucleotide probe to the surface of a gold matrix via a trithio-substituted adamantane derivative; (2) putting the two or more oligonucleotide probes at an environment suitable for DNA hybridization reaction, and adding target DNA for the hybridization reaction; and (3) during the hybridization reaction or after the hybridization reaction is finished, detecting the hybridization reaction system and correlated characteristic parameters of the product formed during the hybridization reaction, so as to detect the target DNA. According to the method, the trithio-substituted adamantane derivative is employed for connecting the oligonucleotide probe and the gold matrix, so that the oligonucleotide probe is chelated with the metal surface via three sulfur atoms, and extremely high metal surface coating stability (such as chemical, optical, temperature, biological, electrochemical and electrical stabilities) is obtained, and thus the smooth performing of correlated experiments are facilitated, and the accuracy of correlated experiment results is improved.
Description
Technical field
The present invention relates to a kind of biomolecule detecting method, particularly a kind of non-marked gene mononucleotide polymorphism (Single Nucleotide Polymorphism, SNP) detection method.
Background technology
Industry has developed multiple by biomolecules is connected in to golden nanometer particle at present, the golden nanometer particle on platinum surface, the surface of the material such as gold electrode, gold nanometer film, builds the technology of biological detection reagent kit, sensor or 3-D nano, structure material then.In existing these technology, biomolecules and gold copper-base alloy be connected normally that single or multiple sulfydryls in biomolecules realize the strong absorption of gold surface by being connected to, but because of sulfydryl poor chemical stability, very easily be subject to oxygen, illumination effect and degrade, and produce by product, the precision of the carrying out of interfere with subsequent reaction or impact test then.
For example, for existing SNP detection method, it is by oligonucleotide probe being connected on the matrixes such as golden nanometer particle, gold thin film mostly, again by observing the combination situation of these oligonucleotide probes and target dna realize, but due to aforesaid defect, often cause detected result not satisfactory.
Summary of the invention
In view of deficiency of the prior art, main purpose of the present invention is to provide a kind of non-marked SNP detection method.
For achieving the above object, the present invention has adopted following technical scheme:
A non-marked SNP detection method, comprising:
(1) provide two or more oligonucleotide probes, and wherein at least one oligonucleotide probe is connected to auri surface by trithio adamantane derivative;
(2) these two or more oligonucleotide probes are placed in to the environment that is suitable for carrying out hybridization, and add target dna to carry out hybridization;
(3), in hybridization process and/or after hybridization completes, by the correlated characteristic parameter of the product of hybridization system and/or hybridization formation is detected, realize the detection to target dna.
As one of comparatively preferred embodiment, step (1) comprising: two or more oligonucleotide probes are connected to auri surface by trithio adamantane derivative respectively, and described auri body comprises golden nanometer particle.
Further, the method comprises:
(2) two kinds of oligonucleotide probes are placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and add respectively target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have above and these two kinds of non-matching sites of oligonucleotide probe DNA standard substance and with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, and carry out hybridization;
(3) to by described target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have one above with the DNA standard substance in these two kinds of non-matching sites of oligonucleotide probe and detect respectively with the fusing point that these two kinds of oligonucleotide probes react the hybridization product forming with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, and determine the corresponding relation between the quantity in described non-matching site and the fusing point of described hybridization product, and then realize the detection to target dna.
As one of concrete application scheme, the method that detects described hybridization product fusing point in step (3) comprises: described hybridization product is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, adjust envrionment temperature, and real-time absorbance spectrum variation with temperature situation, and then record the fusing point of described hybridization product.
As one of comparatively preferred embodiment, step (1) comprising: a kind of oligonucleotide probe is connected to auri surface by trithio adamantane derivative respectively, and described auri body comprises gold thin film chip.
Further, step (2) comprising:
I. a kind of oligonucleotide probe being connected on gold thin film chip is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and adds target dna to carry out hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
Ii. another kind of oligonucleotide probe is added in the hybrid reaction system of step I formation, and proceed hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored.
As one of comparatively preferred embodiment, the method comprises:
(2)
I. a kind of oligonucleotide probe being connected on gold thin film chip is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and add respectively target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have above and these two kinds of non-matching sites of oligonucleotide probe DNA standard substance and with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, carry out hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
Ii. another kind of oligonucleotide probe is added in the hybrid reaction system of step I formation, and proceed hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
(3) determine the corresponding relation between described SPR spectrum parameter variation and the quantity in described non-matching site, and then realize the detection to target dna.
Particularly, described trithio adamantane derivative has following structural formula:
Wherein, R comprises any group.
Further, described trithio adamantane derivative is at least selected from any in following compound:
Wherein,
for a minute sub-connection interval, Y comprises any one or the two or more crossbreds in sugar, medicine, amino acid, polypeptide, Metabolic Intermediate, nanometer construct.
Compared with prior art, the present invention at least tool has the following advantages: by connecting oligonucleotide probe and auri body with trithio adamantane derivative, make oligonucleotide probe system to metallic surface, carry out chelating by three sulphur, can obtain the coated stability in high metallic surface and (include but not limited to chemical stability, optical stability, temperature stability, biologically stable, electrochemistry and electrical stability), thereby be beneficial to carrying out smoothly of correlation test, and promote the tolerance range of correlation test result.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that the present invention typical case implements one of embodiment;
Fig. 2 a is two the schematic diagram that the present invention typical case implements embodiment;
Fig. 2 b is three the schematic diagram that the present invention typical case implements embodiment;
Fig. 2 b is four the schematic diagram that the present invention typical case implements embodiment;
Fig. 3 is five the schematic diagram that the present invention typical case implements embodiment;
Fig. 4 a is the schematic diagram of the present invention's one exemplary embodiments;
Fig. 4 b is the abosrption spectrogram of the present invention's one exemplary embodiments;
Fig. 5 a is the temperature-optical density collection of illustrative plates that utilizes the stable golden nanometer particle probe detection of a target DNA (complete complementary) of trithio diamantane oligonucleotide in one embodiment of the invention;
Fig. 5 b is the temperature-optical density collection of illustrative plates that utilizes the stable golden nanometer particle probe detection of a target DNA (1 base is non-matching) of trithio diamantane oligonucleotide in one embodiment of the invention;
Fig. 6 a is the temperature-optical density collection of illustrative plates that utilizes the stable golden nanometer particle probe detection of a target DNA (complete complementary) of trithio diamantane oligonucleotide in one embodiment of the invention;
Fig. 6 b utilizes the abosrption spectrogram of the stable golden nanometer particle probe detection of a target DNA (1 base is non-matching) of trithio diamantane oligonucleotide under differing temps in one embodiment of the invention;
Fig. 7 a is six the schematic diagram that the present invention typical case implements embodiment;
Fig. 7 b be the present invention typical case implement embodiment six in the spectrogram of differential responses time.
Fig. 8 is the process flow sheet of the present invention's one exemplary embodiments.
Embodiment
First the present invention provides a kind of 2,4, mix-[3.3.1.13 of 9-trithio, 7] tristane-7-carboxylicesters (also can called after " trithio adamantane derivative "), utilize its three contained sulphur to carry out chelating to metallic surface, obtain the high coated stability in metallic surface, and by being applied to being connected of biology, chemical molecular or group and gold copper-base alloy, can effectively promote the effect of all kinds of reactions that realize based on this connection, as, the efficiency of reaction, accuracy of result etc.
As one of application scheme wherein, the present invention can be applied to gene sequencing, by the double-helical thermostability of analyzing DNA, detects the method for individual gene gene order variation in a 20-30 base DNA sequence dna.The method (SNP detection) has been widely used in the diagnosis in heredopathia, forensic medical examination, and the aspects such as evaluation of sex pheromone.
Consult Fig. 1 and Fig. 2 a-2b, aforesaid this exemplary embodiment can comprise: the oligonucleotide molecules surface anchor adhering in the gold surface of utilizing trithio diamantane to detect as oligonucleotide, and can prove the reliability of this method and the simplicity of using by uv-visible absorption spectra (UV-vis) and surface plasma resonance optical spectrum (SPR).
Below in conjunction with some embodiment and accompanying drawing, technical scheme of the present invention is further described.
It is pointed out that the common agents adopting in following embodiment is all purchased from Sigma Aldrich company and Themo Fisher, and directly use.The gold nanoparticle of 13 nanometers that citric acid is stable is prepared according to known references.Oligonucleotide is to buy from Integrated DNA Technologies customization.The all solvents that use are SILVER REAGENT, and distill before use.For the resistance of the ultra-clean water of Nanopure (Milli-Q system) of all experiment higher than 18M Ω.The reaction of all air and moisture-sensitive is carried out under drying nitrogen.On Varian Gemini300 spectrograph, record 1H NMR (300 megahertz) and
13cNMR composes (75 megahertz).Infrared spectra is collected at Nicolet NEXUS870FT-IR spectrograph.ATP-IR is equipped with a Thunderdom ATR annex on aforesaid device.A Japanese Shimadzu UV1800 spectrophotometer of being furnished with TCC240A temperature control system is used to UV-Vis uv-vis spectra and DNA melting curve.Electrospray Ionization Mass Spectrometry (ESI-MS) is to obtain on a Bruker Esquire liquid chromatography (LC)-ion trap mass spectrometry interlock instrument.Self-defined built-in four-way flow-through cell is carried out in SPR measurement, and spr sensor, based on Wavelength demodulation (1 wavelength moves the absorption of correspondence～150pg/mm2 surface coverage biomolecules).Transmission electron microscope image is taken with JEOL JEM1230 transmission electron microscope.
Certainly; content according to this specification sheets; those skilled in the art also can expect adopting alternative aforementioned agents or the equipment such as other is commercially available or self-control approach obtains classes of agents or testing installation easily, so it should not form any restriction to the claimed scope of the present invention.
The synthetic route of the present embodiment can be consulted Fig. 8, and wherein, the reaction conditions of step a-e is as follows: (a): ozone, methylene dichloride ,-78 ℃, dimethyl thioether; (b): thiophosphoric anhydride and aluminium sesquioxide reflux in acetonitrile; (c); Lithium hydroxide is at tetrahydrofuran (THF), the mixture (THF:MeOH:H of first alcohol and water
2o=3:3:1), reflux, then adds 6 equivalent hydrochloric acid neutralizations; (d) 6-bromine hexanol, Anhydrous potassium carbonate, diformamide; (e) 2-nitrile ethyl-N, N-di-isopropyl protochloride phosphamide, diisopropylethylamine, methylene dichloride.
Further, aforementioned synthetic route can by as follows more specifically embodiment realize:
Synthesizing of 2,4,9-trithio diamantane-7-carboxylate methyl ester (compound 2):
The ice bath (78 ℃) of a dry ice and acetone freezing mixture cooling three neck round-bottomed flasks in (500mL), add methyl-2, the anhydrous methylene chloride solution (150mL) of 2-diallyl-penta-obtusilic acid (1,3.00 gram of compound, 15.4 mmoles); Three neck round-bottomed flasks are furnished with the prolong of dry ice and acetone freezing mixture, mechanical stirrer, ozone inlet pipe.Stir after 15 minutes, allow ozone gas (2-5% oxygen partial pressure, is produced by ozonizer) bubbling pass into the yellow solution of clarification, until color change is sky blue, labeled reactant completes.The logical nitrogen of bubbling is removed blue excessive ozone, until it becomes colourless and lasting 5 minutes.Again dimethyl thioether (4 milliliters, 61.6 mmoles) is dropwise joined in reaction mixture, then remove ice bath, container is also stirred and spent the night from being naturally warmed up to room temperature.Then solvent is under reduced pressure removed, obtained yellow sticky solid, then it is dissolved in acetonitrile (100mL) again.Under agitation, slowly add thiophosphoric anhydride (7.53 grams, 16.9 mmoles) and alkali alumina (16.0 grams), mix to produce slurries, and by mixture back flow reaction 25 hours.Then reaction mixture is filtered, with warm ethyl acetate rinse filtration under diminished pressure liquid, obtain crude product, used flash chromatography post separated, wherein elutriant adopts the gradient of the 5%-25% of ethyl acetate and hexane.And then obtain 2,4,9-trithio diamantane-7-carboxylate methyl ester (1.15 grams, productive rate 30%).Corresponding characterization data is as follows:
1h NMR δ: 2.90 (6H, d, CH
2), 3.74 (s, 3H, OCH), 4.32 (w, 3H) PPM; FTIR (ATR): 1723 (C=0), 2941 (fatty CH), 2900 (aliphatic CH) cm
-1place.
Synthesizing of 2,4,9-trithio diamantane-7-carboxylic acid (compound 3):
In round-bottomed flask, add (0.575 gram of compound 2,2.31 mmoles) with (0.506 gram of anhydrous lithium hydroxide, 21.1 mmoles), and add sodium lauryl sulphate and be 3:3 by volume ratio: the mixing solutions of 1 tetrahydrofuran (THF), methyl alcohol and water, and reflux and spend the night.Then by mixture acidifying (being adjusted to pH with concentrated hydrochloric acid is 2), and make it cooling, the refrigerator overnight of 4 ℃, by solution filter, and with icy water washing, obtain lurid product 2,4,9-trithio diamantane-7-carboxylic acid (0.48 gram, 89%).Corresponding characterization data is as follows:
1h NMR δ: 2.93 (6H, d, CH2), 4.38 ((wide), 3H) PPM; Infrared (ATR): 1710 (C=0), 2920 (aliphatics CH), 2933 (aliphatics CH) 2700-3200 (wide, OH) cm
-1place.
3,2,4,9-trithio diamantane-7-carboxylic acid acid oneself-1-alcohol (compound 4) synthetic:
(0.30 gram of compound 3,1.2 mmoles) and (0.53 gram of Anhydrous potassium carbonate, 3.8 mmoles) be dissolved in anhydrous dimethyl formamide (6mL), under nitrogen atmosphere by solution stirring 30 minutes, then by (0.184 milliliter of 6-bromine hexanol, 1.41 mmoles) dropwise join in reaction mixture, then at room temperature stir and spend the night.By TLC, monitor and reacted.After having reacted, reducing solvent evaporate to dryness under vacuum, aforementioned flash chromatography post partition method (ethyl acetate/hexane, 1:3, volume ratio) purifying for crude product, obtains product oily or liquid crystal shape (0.28 gram of productive rate 68%).Its characterization data is as follows:
1h NMR δ: 2.90 (q, 6H, CH
2), 4.33 ((broad peaks)), 3H, CH), 3.66 (t, 2H, OCH
2), 4.14 (t, 2H, OCH
2), 1.68 (2H m, CH
2), 1.59 (m, 2H, CH
2), 1.41 (m, 4H, CH2) PPM.13C?NMRδ：175.20，65.37，62.87，41.36，40.07，38.63，32.73，28.69，25.91，25.59。Infrared: 1725 (C=0), 2934 (aliphatics CH) 2859 (fatty CH), 3323-3500 (wide, OH) cm
-1place.
Synthesizing of 2,4,9-trithio diamantane-7-carboxylic acid hexane phosphoramidite (compound 5):
Compound 4 (0.23 gram, 0.667 mmole) is dissolved in anhydrous methylene chloride (10mL), and adds diisopropyl ethyl amine (0.2 milliliter, 1.16 mmoles), under nitrogen atmosphere, stirring.Then mixture is placed on ice bath and is cooled at 0-2 ℃, continue to stir, and by syringe, dropwise add (0.152 milliliter of 2-cyanoethyl di-isopropyl protochloride phosphamide simultaneously, 0.84 mmole), after adding, reaction mixture is removed from ice bath, and at room temperature stirred 3 hours.By TLC, monitor reaction process.After having reacted, by sodium hydrogen carbonate solution for product (0.5M, 2 * 10 milliliters), collect organic phase, and reduction, the crude product obtaining, by the hexane 5%-25% gradient of ethyl acetate and 1% triethylamine, through aforementioned flash chromatography column separating purification.Obtain oily product (0.186 gram, 52%), its characterization data is as follows:
1h NMR δ: 4.33 ((broad peaks)), 3H, CH), 4.13 (t, 2H, OCH2), 3.81 (m, 2H, NCH), 3.61 (m, 4H, OCH2), 2.90 (d, 6H CH2), 2.65 (t, 2H, CH2CN) 1.64 (m, 4H, CH2), 1.40 (m, 4H, CH2), 1.18 (d, 12H, CH3) PPM.13C?NMRδ：175.05，119.5，65.17，63.27，45.95，45.88，41.36，39.98，38.55，30.51，28.57，25.66，25.47，23.69，22.48PPM。Infrared: 1719 (C=0), 2253 (CN), 2950 (aliphatics CH), 2929 (aliphatics CH), cm
-1.ESI-MS: calculated value (M+) 557.75 finds 557.5.
Synthetic trithio diamantane oligonucleotide:
In the present embodiment, be to use phosphoramidite solid phase oligonucleotide synthesis method to obtain required oligonucleotide chain, consult Fig. 1, it usings trithio diamantane phosphoramidite (compound 5) as a last base.Then by go protection and the purification process of standard, obtain the required oligonucleotide strand that end is trithio diamantane, the strand that the method can be used in DNA and RNA synthesizes.
And based on this trithio diamantane oligonucleotide, can design molecular probe test kit.One, more specifically in application examples, can adopt sandwich assay, that is: selected target sequence (30 bases) is divided into two sections, the strand DNA (desoxyribose nucleic acid) of the complementary sequence of synthetic two strands (first and second) respectively, each is by 15 based compositions.
The preparation of the coated nano Au particle of trithio diamantane oligonucleotide:
By above-mentioned trithio diamantane oligonucleotide and mean diameter, be at room temperature hatching 16 hours with the stable golden nanometer particle of citric acid (about 17nmol) in 13 nanometers, wherein the concentration of trithio diamantane oligonucleotide is 3.5 micromoles, obtain the aqueous solution of the gold nano-probe of oligonucleotide, then make to contain in this solution 0.1M NaCl, and add the 10mM phosphate buffered saline buffer of pH7.4, standing 48 hours.By solution at 20000g centrifugal 30 minutes, isolated nano particle, in the bottom of centrifuge tube, was the red precipitate of oily, and superfluous oligonucleotide is stranded in filtrate.Isolated nano particle is suspended in to the NaCl containing 0.1M again, in the phosphatic damping fluid of 10mM (pH7.4), repeated centrifugation and redispersion (maximum twice), be finally suspended in the damping fluid (pH7.4) containing 0.3M NaCl, 10mM phosphoric acid salt and 0.01% sodiumazide again.In the SNP method of inspection, detected object is cold ssDNA sequence, in this case 30 bases.The design of probe is by these 30 base pairs in two, thereby designs trithio diamantane oligonucleotide probe first, and its end is complementary with the scDNA of 15 bases and half ssDNA of detected object.The end of trithio diamantane oligonucleotide probe second is with other 15 bases, and second half ssDNA is complementary.Probe first and the coated nanoparticle of probe second, the visible absorbance peak of its feature is in 530 nanometers.
DNA probe based on nano Au particle and target oligonucleotide hybridization and fusing point analysis:
The method that cold SNP detects is that coated golden nanometer particle (first and second) and the target sequence of the strand DNA (desoxyribose nucleic acid) of two complementary sequences mixed in solution, in the water-bath of 55-60 ℃, heat 5 minutes, and at room temperature standing, until there is particles aggregate (approximately 2
3 hours).By absorbance spectrum, to the variation of temperature and obtain " fusing point " of this condensed system, the method can accurately measure the variation (SNP) of single base pair.
Consult Fig. 5 a-Fig. 6 b, wherein use the stable golden nanometer particle probe of trithio diamantane oligonucleotide to survey one by the target sequence of 30 bases: A figure is the first and second probes and target sequence complete complementary, B figure is the first and second probes and the complete incomplementarity of target sequence, and C is the test that the first and second probes and target sequence have not complementary target sequence of base to hybridize.
Because 5 ' end of the oligonucleotide chain of these two kinds of probes has trivet anchor, target alignment, and with head, the probe in the mode of tail (Fig. 1) is hybridized.The three-dimensional network of an expansion, functionalized a plurality of oligonucleotide, in red (524 nanometer), bluish pink (569 nanometer).
By measuring the absorbancy at 650nm place, as the function of temperature, the aggregate of the nanoparticle of the temperature of fusion curve of drafting, melt temperature above (Tm) (Fig. 4) shows distinctive sharp-pointed transition.This temperature of fusion, corresponds to 61 ℃, is the thermal dissociation due to the target dna of the DNA functionalization golden nanometer particle probe hybridization from complementary.
In the unmatched target sequence of 1 base pair detects, temperature of fusion has declined approximately 20 ℃.The nanoparticle that the unmatched target of other 1 base pair forms is assembled TM and is observed 41 ℃ (Fig. 5).On unmatched basis on the basis that this minimizing can be explained in melt temperature, can not form hydrogen bond nanoparticle probe and corresponding alkali, cause unsettled impact, form incomplete duplex.This incomplete duplex forms to assemble to form from complementary target at the less heat energy of needs compares oligonucleotide link polymerization sex change.
Surface plasmon resonance (SPR) spectrum list base non-marked SNP detects
Consult Fig. 7 a, above-mentioned RNA sequence detects also can pass through the realization of surface plasmon resonance (SPR) spectrum, the target of first, the second of oligonucleotide probe and oligonucleotide comprises complete complementary, single base that be unworthy of with non-complementary single strand dna.
Being prepared as follows of the gold thin film chip that trithio diamantane oligonucleotide is coated: by gold thin film chip be immersed in trithio diamantane oligonucleotide solution (10 mmole phosphate buffer solutions, 0.3 equivalent sodium-chlor, pH7.4) in 12 hours.After taking out, use ultra-clean water washing, then pack SPR instrument into.
Hybridization check is by injecting the oligonucleotide solution of 5 microlitres, consults Fig. 7 b and can see that the method has very high specificity.
Below be only concrete exemplary applications of the present invention, protection scope of the present invention is not constituted any limitation.All employing equivalents or equivalence are replaced and the technical scheme of formation, within all dropping on rights protection scope of the present invention.
Claims (9)
1. a non-marked SNP detection method, is characterized in that, comprising:
(1) provide two or more oligonucleotide probes, and wherein at least one oligonucleotide probe is connected to auri surface by trithio adamantane derivative;
(2) these two or more oligonucleotide probes are placed in to the environment that is suitable for carrying out DNA hybridization, and add target dna to carry out hybridization;
(3), in hybridization process and/or after hybridization completes, by the correlated characteristic parameter of the product of hybridization system and/or hybridization formation is detected, realize the detection to target dna.
2. non-marked SNP detection method according to claim 1, is characterized in that, step (1) comprising: two or more oligonucleotide probes are connected to auri surface by trithio adamantane derivative respectively, and described auri body comprises golden nanometer particle.
3. non-marked SNP detection method according to claim 2, is characterized in that, the method comprises:
(2) two kinds of oligonucleotide probes are placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and add respectively target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have above and these two kinds of non-matching sites of oligonucleotide probe DNA standard substance and with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, and carry out hybridization;
(3) to by described target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have one above with the DNA standard substance in these two kinds of non-matching sites of oligonucleotide probe and detect respectively with the fusing point that these two kinds of oligonucleotide probes react the hybridization product forming with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, and determine the corresponding relation between the quantity in described non-matching site and the fusing point of described hybridization product, and then realize the detection to target dna.
4. non-marked SNP detection method according to claim 3, it is characterized in that, the method that detects described hybridization product fusing point in step (3) comprises: described hybridization product is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, adjust envrionment temperature, and real-time absorbance spectrum variation with temperature situation, and then record the fusing point of described hybridization product.
5. non-marked SNP detection method according to claim 1, is characterized in that, step (1) comprising: a kind of oligonucleotide probe is connected to auri surface by trithio adamantane derivative respectively, and described auri body comprises gold thin film chip.
6. non-marked SNP detection method according to claim 5, is characterized in that, step (2) comprising:
I. a kind of oligonucleotide probe being connected on gold thin film chip is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and adds target dna to carry out hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
Ii. another kind of oligonucleotide probe is added in the hybrid reaction system of step I formation, and proceed hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored.
7. non-marked SNP detection method according to claim 6, is characterized in that, the method comprises:
(2)
I. a kind of oligonucleotide probe being connected on gold thin film chip is placed in to the environment that is suitable for carrying out oligonucleotide hybridization reaction, and add respectively target dna, have with the DNA standard substance of the sequence of these two kinds of oligonucleotide probe complete complementaries, have above and these two kinds of non-matching sites of oligonucleotide probe DNA standard substance and with the complete non-complementary DNA standard substance of these two kinds of oligonucleotide probes, carry out hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
Ii. another kind of oligonucleotide probe is added in the hybrid reaction system of step I formation, and proceed hybridization, simultaneously with hybridization process described in SPR spectrum real-time monitored;
(3) determine the corresponding relation between described SPR spectrum parameter variation and the quantity in described non-matching site, and then realize the detection to target dna.
8. according to the non-marked SNP detection method described in any one in claim 1-7, it is characterized in that, described trithio adamantane derivative has following structural formula:
Wherein, R comprises any group.
9. non-marked SNP detection method according to claim 8, is characterized in that, described trithio adamantane derivative is at least selected from any in following compound:
Wherein,
for a minute sub-connection interval, Y comprises any one or the two or more crossbreds in sugar, medicine, amino acid, polypeptide, Metabolic Intermediate, nanometer construct.
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| CN201310485829.0A CN103614459A (en) | 2013-10-17 | 2013-10-17 | Non-labeled SNP detection method |
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| CN106290261A (en) * | 2016-09-28 | 2017-01-04 | 中国科学院苏州生物医学工程技术研究所 | A kind of telomerase activation based on SPR technique test system |
| CN106442421A (en) * | 2016-09-28 | 2017-02-22 | 中国科学院苏州生物医学工程技术研究所 | Telomerase inhibitor screening system based on SPR technology |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106290261A (en) * | 2016-09-28 | 2017-01-04 | 中国科学院苏州生物医学工程技术研究所 | A kind of telomerase activation based on SPR technique test system |
| CN106442421A (en) * | 2016-09-28 | 2017-02-22 | 中国科学院苏州生物医学工程技术研究所 | Telomerase inhibitor screening system based on SPR technology |
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