KR101429193B1 - Nucleic acid aptamer specifically binding to chlortetracycline - Google Patents
Nucleic acid aptamer specifically binding to chlortetracycline Download PDFInfo
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- KR101429193B1 KR101429193B1 KR1020130053046A KR20130053046A KR101429193B1 KR 101429193 B1 KR101429193 B1 KR 101429193B1 KR 1020130053046 A KR1020130053046 A KR 1020130053046A KR 20130053046 A KR20130053046 A KR 20130053046A KR 101429193 B1 KR101429193 B1 KR 101429193B1
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Abstract
Description
The present invention relates to a nucleic acid aptamer which specifically binds to chlorertetracycline.
The present invention also relates to a method for detecting chlorptetracycline using the nucleic acid aptamer, a composition for detecting chlorptetracycline containing the nucleic acid aptamer, and a detection kit.
Tetracycline (TC) antibiotics are currently the most widely used antibiotics. These antibiotics are human and veterinary medicines. They are generally used in the treatment of infections caused by bacteria and they are injected directly into the animals in the form of feed or directly administered to prevent infection of fish or marine products.
However, residual tetracycline or its analogue present in animal feed residue or excreta is now absorbed in soil or water, and can be affected by various pathways in the environment, which is problematic. In particular, tetracycline residues in honey, milk, and eggs have already been reported in several studies.
In addition, when a concentrated aquatic product in which antibiotics such as a tetracycline compound is remained is ingested for a long period of time, resistance to the living body may occur, which may cause side effects such as impaired immunity. In other words, if antibiotics abuse on animals leads to general food poisoning bacteria becoming "multidrug resistant bacteria" (super bacteria), common antibiotics will not be able to cure food poisoning, and vicious cycle of administering more virulent antibiotics to heal Is repeated. Therefore, it is necessary to develop a method for detecting residual antibiotics to ensure the safety of food as well as to prevent misuse of antibiotics.
Aptamers are single stranded DNA or RNA structures with high specificity and affinity for a particular target. Aptamer has a much higher affinity for target, higher heat stability, and can be synthesized in vitro than antibodies, which are used in the field of sensors, so it is a cumbersome process to produce antibodies by injecting an antigen into an animal It is possible to omit it and it is superior in terms of cost, there is no restriction on the target material, and aptamers for various targets such as biomolecules such as proteins and amino acids, small organic chemicals such as environmental hormones and antibiotics, and bacteria can be synthesized. Thus, aptamers are very suitable for introduction into trace amounts of residual antibiotics, and can be applied to the detection of other specific substances using nano-bio technology. Due to the characteristics of the aptamer specifically binding to the target substance, a lot of research has been conducted recently for the purpose of using aptamer as a new drug development, drug delivery system, and biosensor. However, most of the existing aptamer development goals were mostly for the development of biosensors for medical diagnosis and the development of new drugs targeting target substances of diseases.
The existing method of detecting residual antibiotics is a method of preparing different test solutions according to the components in which residual antibiotics are dissolved, extracting and purifying residual antibiotics, and quantitatively measuring them by liquid chromatography. The conventional method is a few test methods according to the dissolved substance, and it is troublesome to prepare test solutions separately. In addition, accurate quantification was difficult because the detection substance may be lost during the extraction and purification process. These low molecular weight harmful substance binding specific aptamers such as residual medicines, antibiotics and environmental hormones can be applied not only as a sample in a water source and a river but also as a technology for detecting and diagnosing residual harmful substances in the body such as human body, Antibiotic detection methods have been proposed only for food or water.
Accordingly, the present inventors have made extensive efforts to overcome the problems of the prior art, and as a result, they have developed a nucleic acid aptamer that is a nucleic acid construct having a specifically high affinity to chlorotetracycline, one kind of antibiotic substance. Further, it has been confirmed that residual antibiotic substances can be effectively detected from food, a water supply source, a human body, and the like by producing a composition comprising a nucleic acid aptamer capable of specifically binding to chloretetracycline and a derivative thereof, .
One aspect of the present invention is to provide a nucleic acid aptamer that specifically binds to chlorertetracycline.
Another aspect of the present invention is to provide a method for detecting chlorotetracycline using the nucleic acid aptamer, a composition for detecting chlorptetracycline containing the nucleic acid aptamer, and a detection kit.
One aspect of the present invention provides a nucleic acid aptamer that specifically binds to chloretetracycline, comprising a motif consisting of a base sequence selected from the group consisting of GCCCG, GCG, GTGG, and TGTGCT.
The nucleic acid aptamer of the present invention refers to a small single stranded oligonucleotide capable of specifically recognizing a target substance with high affinity. At this time, when the aptamer is RNA, T can be recognized as U in the base sequence.
In the present invention, chlor tetracycline is a tetracycline compound, the molecular formula is C 22 H 23 ClN 2 O 8, and a molecular weight is 478.88 g / mol. The chlorotetracycline can be obtained from nature, or can be obtained by chemical synthesis or recombinant methods, but is not limited thereto. In addition, the chloretetracycline includes a salt or a derivative thereof.
The aptamer comprises motifs of GCCCG, GCG, GTGG or TGTGCT, preferably the aptamer may comprise a nucleotide sequence of any one of SEQ ID NOS: 9 to 13, more preferably any of SEQ ID NOS: 4 to 8 And may consist of a single nucleotide sequence.
The number of total nucleotides constituting the nucleic acid aptamer may be 15 to 200 nts, preferably 15 to 80 nts. When the total number of nucleotides is small, chemical synthesis and mass production are easier, The advantage in terms of size is also great. In addition, the chemical modification is easy, the in-vivo stability is high, and the toxicity is also low. In addition, each nucleotide contained in the nucleic acid aptamer may include one or more chemical modifications that are the same or different. For example, at the 2 'position of the ribose, a nucleotide in which the hydroxyl group is substituted with an arbitrary atom or group Lt; / RTI > Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom or an -O-alkyl group such as -O-CH 3 , an -O-acyl group such as -O-CHO, 2 ). ≪ / RTI >
In addition, the aptamer may include a nucleic acid sequence having 90% or more and less than 100% homology with the nucleic acid sequence constituting the aptamer. A nucleic acid sequence having 90% or more and less than 100% of homology is a nucleic acid sequence having one or more nucleotides added, deleted, or substituted, and having 90% or more and less than 100% it means.
Further, another aspect of the present invention provides a composition for detecting chloretetracycline comprising the nucleic acid aptamer.
The nucleic acid aptamer of the present invention exhibits high affinity and specificity to chloretetracycline by specifically binding to chloretetracycline, so that residual antibiotic substances can be effectively detected from foods, water sources, human bodies, and the like.
Further, another aspect of the present invention provides a method for detecting chloretetracycline, comprising the step of adding the nucleic acid aptamer to a sample.
In the present invention, a sample is a composition capable of performing analysis, presumably containing or containing a target substance, and is a composition capable of performing analysis, and includes a sample collected from at least one of liquid, soil, air, food, waste, Lt; / RTI > In this case, the liquid may be characterized by water, blood, urine, tears, sweat, saliva, lymph and cerebrospinal fluid. The water may be characterized by precipitation, seawater, lake water, And the waste includes sewage, wastewater, etc., and the animal or plant includes a human body. The plant and animal tissues may include tissues such as mucous membranes, skin, epidermis, hair, scales, eyes, tongue, cheeks, hooves, beak, snout, feet, hands, mouth, nipple, ear and nose.
The added nucleic acid aptamer may be 0.1 nM to 100 nM, preferably 0.1 nM to 10 nM, more preferably 1 nM to 7 nM.
Further, another aspect of the present invention provides a kit for detecting chloretetracycline comprising the nucleic acid aptamer.
The kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partly or wholly made of plastic, glass, paper, foil, / RTI > The container may be fitted with a cap which is initially part of the container or can be fully or partially detachable, which may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which is accessible to the contents by the injection needle. The kit may include an external package, and the external package may include instructions for use of the components.
The nucleic acid aptamer according to the present invention exhibits high affinity and specificity to chloretetracycline by specifically binding to chloretetracycline, so that residual antibiotic substances can be effectively detected from foods, water sources, human bodies, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing binding characteristics of chlorertetracycline (CTC) -specific aptamers. A is the binding assay result of selected aptamers CTC6, CTC8, CTC11, CTC17, and CTC18 using ELAA. The ssDNA library was used as a negative control. B is the saturation curve and dissociation constant (K d ) according to ELAA.
Fig. 2 is a second-order structural model of the selected aptamers predicted with m- fold.
3 is a diagram showing the results of indirect competition (ic) -ELAA. A shows a calibration curve for CTC using different concentrations of aptamer CTC8. B is the competition curve plotted with 5 nM CTC8 in buffer or milk.
Figure 4 shows the cross-reactivity of 5 nM aptamer CTC8 to TC, OTC and CTC.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example 1: Reagents and devices
(BTC), 1,1'-carbonyldiimidazole, tetracycline (TC), oxytetracycline (OTC), hydrochloride of chlortetracycline (CTC), bovine serum albumin Horseradish peroxidase (HRP), 3,30,5,50-tetramethylbenzidine (TMB) liquid substrate, albumin (OVA) from egg whites and Buffer constituents were purchased from Sigma (USA). The HRP-streptavidin conjugate was purchased from Thermo Scientific (France). The ssDNA random library, primers (aptF, aptR, aptRBioT) and biotinylated aptamers were synthesized by Bioneer (Korea). Taq DNA polymerase and dNTP were purchased from GeneAll (Korea). Milk was purchased at the supermarket.
ELAA (enzyme-linked aptamer assay) was performed on Maxisorp polystyrene microtiter plates (NUNC, Denmark). Enzyme activity was detected at 450 nm using Spectra max plus 384 (Molecular Device, USA). PCR amplification was performed in an automated DNA thermocycler (AxyGen, USA). The concentration of ssDNA or dsDNA was measured using Nanodrop (ASP-2680, ACTgene Inc., USA).
Example 2: Changed ELAA Using CTC Specific Of app tamer Selection and sequencing
To select an aptamer specifically binding to CTC, a random ssDNA containing 40 random nucleotides and flanked with an invariant primer annealing portion: 5'-CGTACGGAATTCGCTAGC-N40-GGATCCGAGCTCCACGTG-3 '(SEQ ID NO: 1) The library was used as an initial pool. Three different primers: aptF 5'-CGTACGGAATTCGCTAGC-3 '(SEQ ID NO: 2), aptR 5'-CACGTGGAGCTCGGATCC-3' (SEQ ID NO: 3) and aptRBioT (same as 5'biotin aptR) were used for PCR amplification. Prior to use, was dissolved in a random DNA library pool of 11.25 ug in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 5 mM KCl, 5 mM MgCl 2, pH 7.2) in 100 uL denaturation at 95 ℃ for 10 minutes denatured, rapidly cooled at 4 [deg.] C for 5 minutes and placed in RT for 5 minutes.
Screening of the aptamer for detecting chloretracycline was performed by the ELAA method.
The CTC-BSA complex was synthesized according to the conventional process modified using the EDC coupling method. Total 1 mM CTC was dissolved in 30 mL of phosphate buffered saline buffer (PBS; 1.47 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4) Sodium chloroacetate was slowly added to the solution. The reaction mixture was stirred for 10 hours at room temperature (RT, 25 < 0 > C), precipitated and filtered. CTC-COOH was dried at 60 ° C and confirmed by using a spectrophotometer. Subsequently, 200 uM CTC-COOH was dissolved in 2 mL of N-dimethylformamide, 5 uM BSA was dissolved in 10 mL of PBS (100 mM, pH 8.0), 100 mM EDC was added to 0.5 mL of distilled water ≪ / RTI > The total reaction mixture was stirred at 4 < 0 > C for 4 hours and then kept overnight at 4 < 0 > C. Dialysis was performed using a 10,000 MWCO dialysis cassette (Slide-A-Lyzer, Pierce) for 24 hours to remove unbound CTC. Since unbound CTCs interfere with the spectrometer, reliable results were obtained only after dialysis of the complex. The resulting CTC-BSA complex was identified using a spectrophotometer and stored at -20 ° C for future use.
The specific sequence of the ELAA method is as follows. For the first time, in the coating process, the CTC-BSA complex or BSA was adsorbed in the wells of a microtiter plate under 50 mM carbonate buffer at pH 9.6. The coating volume was 100 uL / well of 10 ug / mL CTC-BSA complex or BSA solution, and the plates were incubated overnight at 4 캜. The cultured plates were coated with 200 uL / well of 1% (w / v) OVA solution (blocking buffer) and prepared in binding buffer for 60 min at 37 ° C. The denatured DNA library pool was then added to the BSA-coated wells to remove candidates that could non-specifically bind to BSA. Unbound DNA was collected and added to CTC-BSA coated wells at 37 < 0 > C for 60 minutes. After incubation, the unbound or weakly bound DNA was removed in three washing steps with PBST. Bound DNA was eluted with 200 uL elution buffer containing Tris-HCl (40 mM, pH 8.0), EDTA (10 mM), urea (3.5 M), and Tween 20 (0.02% Eluted from the coated wells, and ssDNA was recovered. This procedure was repeated three times to elute the bound ssDNA. The eluted oligonucleotides were precipitated using the ethanol precipitation method, and the ssDNA was dissolved in 10 uL of EB buffer (10 mM Tris-HCl, pH 8.5). The eluted ssDNA fractions were analyzed by PCR (1 cycle at 95 ° C for 5 minutes, 35 cycles at 95 ° C for 30 seconds, 72 ° C for 30 seconds, and 1 cycle at 72 ° C for 5 minutes) Was amplified using a biotinylated reverse primer (aptRBioT), which allowed subsequent purification of the aptamer strand using alkaline denaturation in Dynabeads streptavidin M-280. The final amount of ssDNA in each round was determined using Nanodrop. After 12 rounds, ssDNA was amplified using unmodified aptF and aptR primers and cloned in TOPO PCRII (Invitrogen). We sequenced 11 inserted candidate app trams.
As a result, five of the 11 aptamers inserted into the clones were identified and screened for CTC binding affinity. Five selected aptamers were designed as CTC6, CTC8, CTC11, CTC17 and CTC18. The sequence numbers and sequence information of the 5 aptamers are shown below.
Among these candidates, it was confirmed that the four aptamers CTC6, CTC8, CTC17 and CTC18 exhibited good binding affinity to CTC. It was confirmed that the aptamers CTC6, CTC8, CTC17 and CTC18 had a higher binding capacity to CTC than the other aptamers (FIG. 1A).
The sequences for the variable 40-nucleotide (N 40 ) positions of five aptamers using ClustalW were analyzed and the results are shown in Table 2 below.
As can be seen in Table 2 above, it was found that the aptamer contains a highly conserved sequence (GCCCG or GCG) and a number of consensus sequence motifs (GTGG or TGTGCT). This conserved sequence and the common sequence motif are palindromic sequences, which are thought to influence the specific binding of aptamers.
The secondary structure of these app tamers was also specified using an m-fold program (Fig. 2). The conserved sequence and common sequence motifs of the app tamer were located at the stem and ring sites of each aptamer (Table 2 and Figure 2). Despite the differences in the N40 variable regions of the aptamer, the secondary structure of these aptamers had a very similar structure. In particular, CTC6 and CTC8 had very similar structures to each other in comparison with other aptamer structures. However, these two aptamers differed in their ability to bind to CTC (Fig. 1). These results indicate that highly conserved sequence motifs and secondary structures among the sequences of five aptamers affect the recognition and binding of CTC.
Example 3: Selected Of app tamer CTC Confirmation of binding properties for
The ELAA method was performed to compare the binding affinities of selected aptamers to CTC. BSA-coated well plates were also used to confirm whether aptamers bind to BSA. A random oligonucleotide (Oligo) library was used as a negative control.
Initially, the biotinylated aptamer was heated at 90 ° C for 5 minutes, cooled at 4 ° C for 5 minutes, and placed at RT for 10 minutes. Then, 5 nM of each aptamer was incubated in a well coated with BSA or CTC-BSA in a microtiter plate blocked with 1% (w / v) OVA solution at 37 ° C for 60 minutes. After the binding reaction, streptavidin-HRP (0.5 ug / mL) solution was added and reacted (30 min, 37 ° C). The final step was to add 100 uL of TMB solution and measure absorbance at 450 nm after 30 min (after quenching with the addition of 100 uL / well of 1 NH 2 SO 4 ). Each step was performed with repeated agitation and blocked with light. Also, the wells are rinsed three times with PBST between each step. The assay was performed three times.
To determine the optimal run conditions for CTC and aptamers, different concentrations (1 ug, 5 ug and 10 ug / mL) of the fixed CTC-BSA complex were exposed to various concentrations of biotinylated aptamers. The coatings of the wells were performed using a CTC-BSA complex at 5 ug / mL. The concentration of streptavidin-HRP was 0.5 ug / mL. Binding reactions were performed using a constant quantity of the CTC-BSA complex to the concentration of a row of selected aptamers (1-20 nM).
It was determined aptamer CTC6, CTC8, CTC17 and dissociation constant (dissociation constant) of CTC18 (K d) (Fig. 1B). The binding reaction was carried out as above but using an increased amount of biotinylated aptamer (1-20 nM). The amount of aptamer bound to CTC was determined by absorbance measurement and was converted to% binding. Value and equal to:% (A / A 100) = (A / A 100) × 100, where A is 1 nM to an aptamer positive absorbance values of 20 nM, 100 is A Absorbance value of 20 nM aptamer CTC8. K d was calculated by nonlinear regression analysis.
Figure IB shows a non-linear regression analysis for the combined data. Aptamers was determined by non-linear regression analysis of CTC6, CTC8, CTC17 and the dissociation constant (dissociation constant) (K d) a combination of CTC18 mutator (Fig. 1B). The dissociation constants for the four aptamers were in the nanomolar range. Of these aptamers, CTC8 and CTC18 This showed the best binding affinity, showed a very low K d values (4.2 and 5.6 nM) compared to other aptamers CTC16 and CTC17 (Fig. 1B). The K d values of CTC16 and CTC17 were 10.4 and 13.7 nM (Fig. 1B). The highest avidity CTC8 was used to confirm CTC detection.
Example
4: indirect competition
ELAA
(
indirect
competitive
ELAA
,
ic
-
ELAA
)
CTC
Detection
In an indirect competition assay, the concentration of the aptamer and immobilized antigen is an important factor in determining the sensitivity of the aptamer to the target material. Ic-ELAA depends on the concentration of the fixed CTC-BSA complex and the concentration of the aptamer.
In addition, screening of the blocking solution is necessary to prevent nonspecific adsorption. In general, serum albumin and dry milk are used as blocking solutions in immunoassays. In this assay, two different blocking solutions were found to reduce background interference. Milk powder showed a higher basicity than OVA, because it contains endogenous biotin. Thus, this affects the assay results using the biotin-streptavidin system.
Specifically, competition was performed by addition of non-labeled CTC and biotinylated aptamer. The solution was prepared in binding buffer and the competition time was 60 minutes at 37 < 0 > C. After the competition reaction, the staptabidin-HRP solution was added and reacted. Finally, 100 uL of TMB solution was added and the absorbance was measured as described above. The assay was performed five times. The absorbance values were converted to experimental inhibition values (A / A 0 ) as follows: (A / A 0 ) = (A / A 0 ) × 100 where A is the absorbance value of the competition assay and A 0 is the absorbance value of the non-competitive assay.
As shown in Figure 3A, the concentrations of three CTC8 (5, 10 and 20 nM) were used to analyze the sensitivity to CTC, and the concentration of CTC-BSA coated plate wells was determined using CTC-specific aptamers The concentrations used were the same as those used in the binding assays. A concentration of 5 nM of aptamer was chosen to give optimal sensitivity and sufficient absorbance values for easy detection signals. These results indicate that sensitivity is increased when using a small amount of aptamer.
Figure 3B shows the dose-response curves obtained under optimal conditions with 5 ug / mL of CTC-BSA coated on plate wells and 5 nM of aptamer CTC8. The calibration curve of this aptamer was calculated as follows: (1) Binding (%) of CTC8 in buffer = -15.26 × In [CTC] +21.251, (R 2 = 0.9835); (2) Binding (%) of CTC8 in milk = - 15.84 × In [CTC] + 22.636, (R 2 = 0.9746).
In addition, the LOD and LOQ of aptamer CTC8 having the highest sensitivity to CTC were calculated, and the results are shown in Table 3 below.
(
ug
/ L)
(
ug
/ L)
(Average, %)
(
Linearity
),
R
2
As shown in Table 3, the LOD and LOQ of aptamer CTC8 with the highest sensitivity to CTC were 11.1 and 60.8 ug / L in buffer, respectively. LOD and LOQ in milk were 16.6 and 90.7 ug / L, respectively.
Example 5: CTC For Of app tamer Identification of specificity
In the aptamer assay, it was determined specifically whether the aptamer could selectively respond to the target material. The specificity of the selected aptamer, CTC8, for CTC was evaluated using cross-reactivity. Cross-reactivity was achieved by indirect competition of tetracycline (TC) and oxytetracycline (OTC), structurally similar to CTC, using ELAA.
The 50% B /
(%)
A 1 is Cross concentration, A 2 of the CTC represents the 50% B / B 0 represents a 50% B / B 0 - density, B / B 0 of the reaction in the absence of free reactants, free of the B 0 value in response to the maximum It is the reaction rate of the B value when the reactant is present.
As shown in FIG. 4 and Table 4, CTC8 showed a cross-reactivity of more than 30% for TC and a cross-reactivity for OTC was less than 10%. These results are considered to be related to the common sequence motif (GTGG). However, it is believed that highly conserved sequence motifs (GCG or CGGC) more strongly affect the binding affinity for CTC than GTGG motifs.
Example 6: ic - ELAA And HPLC - UV In milk CTC Detection
The recovery of CTC from milk was performed using two different methods, ic-ELAA and HPLC-UV. Milk samples with various concentrations of CTC (50 ug / L to 400 ug / L) were extracted with McIlvaine buffer. The McIlvaine buffer containing EDTA prevents the chelating material from interdigitating with CTC in milk, which can improve the extraction efficiency for the analyte. Recovery rates of CTC in milk samples were analyzed simultaneously with ic-ELAA and HPLC-UV, and the concentrations of CTC obtained in the two methods were compared.
Specifically, milk samples were purchased at local supermarkets and stored at 4 ° C prior to use. Milk contains a cation, such as protein, fat, carbohydrate and Ca 2 + and Mg 2 capable of forming a chelating complex with a CTC. To remove such material, 5 mL of milk sample was mixed with 5 mL of McIlvaine buffer containing 0.02M EDTA (pH 5.0) and 0.5% (v / v) tree Trifluoroacetic acid was added. The milk samples were then centrifuged at 8,000 rpm at 4 ° C for 20 minutes to remove fat and protein. 1 M NaOH was added to the supernatant to titrate to pH 7.0. The supernatant was filtered through a 0.45 um filter (Corning, Germany) and evaporated at 40 < 0 > C using gentle nitrogen blow-down. After extraction, the precipitate was resuspended in binding buffer. Detection of CTCs in milk samples was verified by ic-ELAA using aptamer CTC8 as described above, and HPLC-UV systems were performed by conventionally known methods for its analysis.
The recovery rates of CTC from the milk samples calculated using ic-ELAA and HPLC-UV are shown in Table 5 below.
(
ug
/ L, mean ± SD).
(%, Mean ± SD)
As shown in Table 5, the recovery of CTC added to milk was 91% to 104% in ic-ELAA and HPLC-UV. In both methods, the recovery rate of CTC in milk was found to be 90% or more.
<110> Hoseo University Academic Cooperation Foundation <120> Nucleic acid aptamer specifically binding to chlortetracycline <130> 1-3 <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> invariable primer annealing part containing 40 random nucleotide <400> 1 cgtacggaat tcgctagcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngg 60 atccgagctc cacgtg 76 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> aptF primer <400> 2 cgtacggaat tcgctagc 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> aptR primer <400> 3 cacgtggagc tcggatcc 18 <210> 4 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC6 full sequence <400> 4 cgtacggaat tcgctagcga aagggacggc ccgtacgatt cagaattctg ccgactgggg 60 atccgagctc cacgtg 76 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC8 full sequence <400> 5 cgtacggaat tcgctagccc acaccaccat gcgttcacgt ggccagtgtg cccgttgtgg 60 atccgagctc cacgtg 76 <210> 6 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC11 full sequence <400> 6 cgtacggaat tcgctagcac ctcggcaagg cgcggactcc aggcggtagg gatgtgctgg 60 atccgagctc cacgtg 76 <210> 7 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC17 full sequence <400> 7 cgtacggaat tcgctagcac aggcgggcgg tcatgactac agcccggaga ccgtccgtgg 60 atccgagctc cacgtg 76 <210> 8 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC18 full sequence <400> 8 cgtacggaat tcgctagcac accgcgacgc gttgtcagtg gctgctatac atgcgcgtgg 60 atccgagctc cacgtg 76 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC6 N40 sequence <400> 9 gaaagggacg gcccgtacga ttcagaattc tgccgactgg 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC8 N40 sequence <400> 10 ccacaccacc atgcgttcac gtggccagtg tgcccgttgt 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC11 N40 sequence <400> 11 acctcggcaa ggcgcggact ccaggcggta gggatgtgct 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC17 N40 sequence <400> 12 acaggcgggc ggtcatgact acagcccgga gaccgtccgt 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer CTC18 N40 sequence <400> 13 acaccgcgac gcgttgtcag tggctgctat acatgcgcgt 40
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| US20050023155A1 (en) | 2000-02-17 | 2005-02-03 | Sawyer Jaymie Robin | Protein and peptide sensors using electrical detection methods |
| KR20090103100A (en) * | 2008-03-27 | 2009-10-01 | 고려대학교 산학협력단 | Twenty one Single-stranded DNA aptamers having high affinity to tetracycline and it's analogues with high specificity and production method thereof |
| KR20130093367A (en) * | 2012-02-14 | 2013-08-22 | 고려대학교 산학협력단 | Nucleic acid aptamer capable of specifically binding to tetracyclines compound and use thereof |
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| KR20090103100A (en) * | 2008-03-27 | 2009-10-01 | 고려대학교 산학협력단 | Twenty one Single-stranded DNA aptamers having high affinity to tetracycline and it's analogues with high specificity and production method thereof |
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