KR101754844B1 - Screening method of DNA aptamer - Google Patents
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- KR101754844B1 KR101754844B1 KR1020150065796A KR20150065796A KR101754844B1 KR 101754844 B1 KR101754844 B1 KR 101754844B1 KR 1020150065796 A KR1020150065796 A KR 1020150065796A KR 20150065796 A KR20150065796 A KR 20150065796A KR 101754844 B1 KR101754844 B1 KR 101754844B1
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Abstract
The present invention relates to a method for screening a DNA aptamer, and more particularly, to a method for screening a DNA aptamer comprising a target substance reaction step of mixing a random nucleic acid library with a target substance to induce binding and a specific separation method after the target substance reaction step A nucleic acid-binding reaction step of removing a nucleic acid not bound to a target substance and recovering a nucleic acid conjugate formed by binding a target substance and a nucleic acid, and a specific separation method for a nucleic acid conjugate in order to remove the weakly bound nucleic acid, A nucleic acid separation step of separating the nucleic acid from the nucleic acid complex finally reassembled after the reassembly step is repeated a plurality of times, and a nucleic acid separation step of separating the nucleic acid from the finally reassembled nucleic acid complex, A DNA aptamer screen that can easily and quickly find DNA It is about.
Description
The present invention relates to a method for screening a DNA aptamer, and more particularly, to a method for screening a DNA aptamer comprising a target substance reaction step of mixing a random nucleic acid library with a target substance to induce binding and a specific separation method after the target substance reaction step A nucleic acid-binding reaction step of removing a nucleic acid not bound to a target substance and recovering a nucleic acid conjugate formed by binding a target substance and a nucleic acid, and a specific separation method for a nucleic acid conjugate in order to remove the weakly bound nucleic acid, A nucleic acid separation step of separating the nucleic acid from the nucleic acid complex finally reassembled after the reassembly step is repeated a plurality of times, and a nucleic acid separation step of separating the nucleic acid from the finally reassembled nucleic acid complex, A DNA aptamer screen that can easily and quickly find DNA It is about.
Aptamer refers to single-stranded DNA or RNA having high affinity and selectivity for a specific target substance. Aptamer is a target DNA or RNA having a higher affinity and selectivity for a target substance than an antibody used mainly in a sensor field for disease diagnosis, biosensor, And can be stored at room temperature for a long period of time, and it is easy to chemically deform. Therefore, there is no need to obtain an antibody by injecting an antigen into an animal, and various advantages . Therefore, a lot of researches have been conducted to use aptamers having a high selectivity and affinity specifically for a target substance to be used for diagnosis, therapeutic agent technology, biosensor, and the like. In order to use the aptamers in diagnostic and therapeutic technologies, biosensors, and the like, it is essential to screen aptamers having a high selectivity and affinity specifically for a target substance. For example, as shown in the following patent documents, A DNA aptamer that specifically binds to the target substance (kanamycin) is screened.
<Patent Literature>
Published patent application No. 10-2012-07077154 (published on 07. 07.10.2010) "DNA aptamer that specifically binds to caramyline"
The conventional method for locating an aptamer specifically binding to a target substance including the above-mentioned patent documents and the like is carried out by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process. (1) binding a nucleic acid library (> 10 15 ) having various forms to a target substance (target substance, protein, cell, peptide, etc.) (3) obtaining a nucleic acid construct bound to the target substance, (4) separating the nucleic acid construct from the target substance, (5) amplifying the nucleic acid construct, ) Is repeated 5 to 15 times more to find an aptamer exhibiting excellent binding force and specificity. However, the existing SELEX - based selection method of the app tamer is very complicated, requires optimum condition, advanced technology, and skilled engineer. In particular, in the process of amplifying the nucleic acid construct of the process (5), the design of the primer, the conditions of the PCR, the amplification of the nucleic acid construct, and the separation of the double stranded DNA into the single stranded DNA are carried out under optimum conditions, complicated processes, There is a disadvantage in that it is not only necessary but also takes a long time.
SUMMARY OF THE INVENTION The present invention has been made to solve the above problems,
It is an object of the present invention to provide a DNA aptamer screening method capable of simply and rapidly identifying a DNA aptamer that specifically binds to a target substance.
In addition, the present invention provides a method of amplifying a nucleic acid having a high affinity and selectivity without requiring a simple and rapid technique, by repeating centrifugation of a nucleic acid conjugate formed by combining a nucleic acid and a target substance, It is intended to provide a DNA aptamer screening method capable of detecting an aptamer.
The present invention also provides a DNA aptamer screening method capable of reacting a recovered nucleic acid with a reverse-sorting substance to remove a bound nucleic acid and recovering a nucleic acid that does not bind to the target substance, thereby further increasing affinity and selectivity for the target substance. .
In order to achieve the above object, the present invention is implemented by the following embodiments.
According to an embodiment of the present invention, a DNA aptamer screening method according to the present invention comprises: a target substance reaction step of mixing a random nucleic acid library with a target material to induce binding; Performing a specific separation method after the target material reaction step to remove a nucleic acid that is not bound to the target substance, and recovering a nucleic acid conjugate formed by binding the target material and the nucleic acid; A step of performing a specific separation method on the nucleic acid conjugate recovered in the step of collecting the conjugate in order to remove the weakly bound nucleic acid to remove the nucleic acid not bound to the target substance to recover the nucleic acid conjugate; And a nucleic acid separation step of separating the nucleic acid from the nucleic acid complex re-recovered in the re-recovery step.
According to another embodiment of the present invention, there is provided a DNA aptamer screening method comprising: a target substance reaction step of mixing a random nucleic acid library with a target material to induce binding; Performing a specific separation method after the target material reaction step to remove a nucleic acid that is not bound to the target substance, and recovering a nucleic acid conjugate formed by binding the target material and the nucleic acid; A nucleic acid conjugate is subjected to a specific separation method in order to remove the weakly bound nucleic acid to remove the nucleic acid that is not bound to the target substance, thereby allowing the nucleic acid conjugate to be recovered; And a nucleic acid separation step of separating the nucleic acid from the finally recovered nucleic acid complex after the re-collection step is repeated a plurality of times, wherein the re-collection step is performed a plurality of times, so that in the first re- A specific separation method is performed on the nucleic acid conjugate recovered in the step 2, and a specific separation method is performed on the nucleic acid conjugate recovered in the immediately preceding re-circulation step in the second and subsequent rejuvenation step.
According to another embodiment of the present invention, the DNA aptamer screening method according to the present invention comprises: an amplification step of amplifying a nucleic acid specifically binding to a target substance by performing PCR on the nucleic acid separated in the nucleic acid separation step; Further comprising:
According to another embodiment of the present invention, a DNA aptamer screening method according to the present invention comprises: a step of reacting a nucleic acid separated in the nucleic acid separation step with a reverse screening substance to induce binding; A nucleic acid recovery step of performing a specific separation method after the reverse sorting material reaction step to remove a reverse sorting material complex formed by binding a reverse sorting material and a nucleic acid and recovering a nucleic acid not bound to the reverse sorting material; .
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the inverse sorting material reaction step and the nucleic acid recovery step are repeated a plurality of times in sequence, And the nucleic acid recovered in the previous nucleic acid recovery step is mixed with the reverse sorting substance to induce binding.
According to another embodiment of the present invention, the DNA aptamer screening method according to the present invention is characterized in that the nucleic acid recovered in the nucleic acid recovery step is used for the target substance reaction step, the conjugate recovery step, .
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the random nucleic acid library is a ssDNA library having a fixed primer region for PCR at both ends and 30 to 50 random bases in the center .
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the centrifugal separation method using a centrifuge is used as the specific separation method.
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the re-screening step is repeated 4 to 39 times.
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the step of reacting the inverse sorting material and the step of recovering the nucleic acid are repeated 2 to 10 times in order.
According to another embodiment of the present invention, in the DNA aptamer screening method according to the present invention, the RPM in the centrifugal separation method using the centrifugal separator is 10 to 100,000.
According to the present invention, the following effects can be obtained by this embodiment.
The present invention has the effect of simply and rapidly identifying a DNA aptamer that specifically binds to a target substance.
In addition, the present invention provides a nucleic acid amplification method and a nucleic acid amplification method, which are capable of repeatedly centrifuging a nucleic acid conjugate formed by combining a nucleic acid and a target substance, and finally recovering the separated nucleic acid, .
In addition, the present invention has the effect of reacting the recovered nucleic acid with a reverse-sorting substance to remove the bound nucleic acid and recovering the unbound nucleic acid, thereby further increasing the affinity and selectivity to the target substance.
1 is a reference diagram for explaining a DNA aptamer screening method according to an embodiment of the present invention.
2 to 4 are reference views showing a secondary structure of a DNA aptamer obtained through a DNA aptamer screening method according to an embodiment of the present invention.
FIGS. 5 to 7 are graphs showing the results of analyzing the binding force between DNA aptamer and E. coli obtained by DNA aptamer screening according to an embodiment of the present invention.
8 is a graph showing the results of analyzing the binding force of DNA aptamer obtained by the DNA aptamer screening method according to an embodiment of the present invention with E. coli and five bacteria.
Hereinafter, a DNA aptamer screening method according to the present invention will be described in detail with reference to the accompanying drawings. Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and, if conflict with the meaning of the terms used herein, It follows the definition used in the specification. Further, the detailed description of known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted. Throughout the specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.
The DNA aptamer screening method according to the first embodiment of the present invention will be described with reference to FIG. 1. The DNA aptamer screening method comprises a target substance reaction step of mixing a random nucleic acid library with a target substance to induce binding, Wow; Performing a specific separation method after the target material reaction step to remove a nucleic acid that is not bound to the target substance, and recovering a nucleic acid conjugate formed by binding the target material and the nucleic acid; A nucleic acid conjugate is subjected to a specific separation method in order to remove the weakly bound nucleic acid to remove the nucleic acid that is not bound to the target substance, thereby allowing the nucleic acid conjugate to be recovered; A nucleic acid separation step of separating the nucleic acid from the nucleic acid complex re-reacted in the re-circulation step; And amplifying the nucleic acid specifically binding to the target substance by performing PCR on the nucleic acid separated in the nucleic acid separation step. A DNA aptamer that specifically binds to the target substance with high affinity can be obtained by cloning and sequencing the nucleic acid amplified in the amplification step.
In the target material reaction step, a random nucleic acid library (DNA library) and a target material (Targets) are mixed to induce binding. The random nucleic acid library has a primer region for PCR at both ends SsDNA library having 30 to 50 random bases in the center is used. Target substances include various substances such as microorganisms, proteins, and viruses, which can be precipitated by centrifugation.
In the step of recovering the complex, a specific separation method is performed after the target material reaction step to remove the nucleic acid that is not bound to the target substance, and the nucleic acid complex formed by the binding of the target substance and the nucleic acid is recovered. Centrifugal separation method using a centrifuge is used. In the centrifugal separation method using the centrifugal separator, RPM is 10 to 100000. In the centrifugal separation method using the centrifugal separator, precipitation, osmotic pressure, filter, column and the like are used do.
In order to remove the nucleic acid bound weakly to the nucleic acid complex, the nucleic acid complex is subjected to a specific separation method (centrifugation method using a centrifuge) to remove the nucleic acid not bound to the target substance, Wherein the re-counting step may be performed one or more times, and when the re-counting step is performed a plurality of times, in the first re-counting step, the nucleic acid conjugate recovered in the recovering step is centrifuged using a principle separator The centrifugation method is carried out for the nucleic acid conjugate that has been re-collected in the immediately preceding re-collection step. When the re-take-off step is performed a plurality of times, it is preferably repeated 4 to 39 times.
The nucleic acid separation step separates the nucleic acid from the nucleic acid conjugate recovered in the re-circulation step. When the re-circulation step is repeated a plurality of times, the nucleic acid is finally separated from the re-circulated nucleic acid conjugate.
In the amplification step, the nucleic acid that specifically binds to the target substance is amplified by performing PCR on the nucleic acid separated in the nucleic acid separation step, and the nucleic acid amplified in the amplification step is cloned and sequenced, A DNA aptamer that specifically binds with affinity can be obtained. In the DNA aptamer screening method, the ssDNA which binds strongly to the target substance can be removed and the weakly bound ssDNA can be removed by repeating the re-sampling step using the centrifugal separation method several times, and finally the recovered ssDNA 1 A DNA aptamer that specifically binds to a target substance with high affinity to the target substance can be obtained by merely amplification, and DNA aptamers can be easily and quickly detected in comparison with the conventional SELEX method which involves several ssDNA separation and amplification steps .
The DNA aptamer screening method according to the second embodiment of the present invention comprises a reaction step of reacting a nucleic acid separated in the nucleic acid separation step between a nucleic acid separation step and an amplification step with a reverse screening substance to induce binding; A nucleic acid recovery step of performing a specific separation method after the reverse sorting material reaction step to remove a reverse sorting material complex formed by binding a reverse sorting material and a nucleic acid and recovering a nucleic acid that is not bound to the reverse sorting material . In the amplification step of the DNA aptamer screening method, the nucleic acid recovered in the nucleic acid recovery step is amplified.
In the step of reacting the inverse sorting material, the nucleic acid separated in the nucleic acid separation step is mixed with a reverse sorting material to induce binding, and the reverse sorting material is a substance different from the target substance and capable of precipitating and separating by centrifugation. , Proteins, and viruses. For example, in the following examples, Escherichia coli is used as a target substance and Bacillus subtilis , Klebsiella pneumoniae , Klebsiella pneumoniae , Enterobacter aerogenes , Staphylococcus, epidermidis ) were used.
In the nucleic acid recovery step, a specific separation method (centrifugation method using a centrifugal separator) is performed after the reverse sorting material reaction step to remove the reverse sorting material combination formed by binding the reverse sorting material and the nucleic acid, And recovering the unreacted nucleic acid. In this case, in the second and subsequent reverse-sorting material reaction steps, the nucleic acid recovered in the previous nucleic acid recovery step is mixed with the reverse-sorting material to form a binding reaction. . When the reverse-sorting material reaction step and the nucleic acid recovery step are performed a plurality of times, it is preferable that the reverse-sorting material reaction step and the nucleic acid recovery step are repeated 2 to 10 times in order. Since the DNA aptamer screening method uses the ssDNA isolated in the nucleic acid isolation step and repeats the inverse sorting material reaction step and the nucleic acid recovery step a plurality of times in sequence, it is possible to remove the ssDNA binding to the reverse sorting material, The selectivity of the material can be further increased.
The DNA aptamer screening method according to the third embodiment of the present invention is characterized in that the nucleic acid recovered in the nucleic acid recovering step between the nucleic acid recovering step and the amplifying step is used for the target substance reaction step, The steps are carried out again in order. In the DNA aptamer screening method according to the third embodiment of the present invention, the target substance reaction step, the conjugate recovery step, the recovering step, and the nucleic acid separation step may be performed using a nucleic acid recovered in the nucleic acid recovery step instead of the prepared random nucleic acid library The same steps as the target substance reaction step, the conjugate recovery step, the re-take step, and the nucleic acid separation step of the DNA aptamer screening method according to the first embodiment described above are performed, and thus a detailed description will be omitted. That is, in the DNA aptamer screening method, the ssDNA having selectivity to a target substance is selected (selection process) through the target substance reaction step, the conjugate recovery step, the reac- tion step and the separation step, and the selected ssDNA is subjected to the reverse screening The ssDNA having the selectivity to the reverse-sorting material is removed through the material reaction step and the nucleic acid recovery step to recover the ssDNA not bound to the reverse-sorting material (inverse selection step), and the ssDNA recovered in the reverse- (SsDNA) having selectivity in the target substance is selected (selection process) and amplified to perform a further step of isolating DNA aptamer having high selectivity for the target substance It is possible. Hereinafter, a DNA aptamer screening method according to a third embodiment of the present invention will be described in more detail with reference to examples. The above-described DNA aptamer screening method of the present invention may be performed after amplification after the screening process (first embodiment), a screening process and a reverse screening process are sequentially performed and then amplified (second embodiment), or a screening process, a reverse screening process, (Third embodiment). This is an example, and it is exemplified by the DNA aptamer screening method such as the selection process, the reverse screening process, the screening process, the reverse screening process, May be followed by various combinations of the sorting process and the inverse sorting process.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are only for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto. In the following, a method of selecting a DNA aptamer that selectively binds to E. coli using E. coli as a target substance has been described. However, the present invention is not limited thereto, and the present invention can be applied to a method for screening proteins, viruses It is possible to select a DNA aptamer that selectively binds to the target substance.
Example 1 Preparation of target substance
The target substance (E. coli) was cultured in a 100 mL NB (nutrient broth) medium until the concentration reached 10 8 CFU / mL, then 1 mL was taken and E. coli was isolated by a centrifuge. The isolated Escherichia coli was washed three times with PBS buffer (pH 7.0) and suspended in binding buffer (1x PBS, 0.1 mg / ml salmon sperm DNA, 1% BSA, 0.05% tween-20) to prepare an Escherichia coli suspension.
Example 2: Preparation of nucleic acid library
A nucleic acid library (ssDNA library) having a fixed sequence region for PCR and 45 arbitrary nucleotide sequences in the center was synthesized at both ends. The ssDNA library is represented by 5-GCAATGGTACGGTACTTCC (SEQ ID NO: 10) -N45-CAAAAGTGCACGCTACTTTGCTAA (SEQ ID NO: 11) -3 and both ends are composed of a fixed base sequence region in which primer pairs are annealed, And an aligned base sequence region (N 45 ). Wherein N 45 generally means that it consists of 45 arbitrary A, T, G, C bases. The ssDNA library was synthesized in Genotech Inc. (Korea).
≪ Example 3 > Selection of ssDNA
1) The Escherichia coli suspension (10 7 cells) prepared in Example 1 and the 10 15 ssDNA libraries prepared in Example 2 were mixed together and reacted at room temperature for 1 hour.
2) Thereafter, the ssDNA not bound to E. coli was removed using a centrifuge, the ssDNA bound to the E. coli was recovered, and the recovered ssDNA bound to the recovered E. coli was centrifuged again using a centrifuge to not bind E. coli The ssDNA is removed and the ssDNA bound to the E. coli is recovered. The ssDNA not bound to the E. coli is removed using the principle separator and the process of collecting the ssDNA bound to the E. coli is repeated ten times.
3) To isolate ssDNA bound to the finally recovered E. coli, the heat was heated at 95 ° C. for 10 minutes, immediately cooled, and maintained at 4 ° C. for 10 minutes to isolate ssDNA bound to E. coli to isolate E. coli ssDNA was selected.
Example 4 Reverse Screening of ssDNA
In order to increase the selectivity of ssDNA selected for Example 3 to Escherichia coli, the selected ssDNA was mixed with a reverse-discriminating substance, ssDNA binding to the reverse-discriminating substance was removed using a centrifugal separator, and ssDNA Was recovered. SsDNA not bound to the recovered reverse-sorting material is mixed with the reverse-sorting material, and centrifuged again using a centrifugal separator to remove the ssDNA bound to the reverse-sorting material and recover the ssDNA not bound to the reverse- SsDNA bound to the reverse-sorting material was removed using the principle separator, and ssDNA not bound to the reverse-sorting material was recovered three times. Examples of the reverse-sorting material include Bacillus subtilis , Klebsiella pneumoniae , Klebsiella pneumoniae ), Enterobacter aerogenes , and Staphylococcus epidermidis were used.
Example 5: ssDNA repeat selection
In order to further increase the binding force and affinity of ssDNA against E. coli, ssDNA which was not bound to the reverse-discriminating substance finally in Example 4 was mixed with E. coli and the procedures of 2) and 3) of Example 3 were repeated.
Example 6 Analysis of the ssDNA Nucleotide Sequence
1) The ssDNAs obtained in Example 5 were amplified by PCR, cloned using a TOPO cloning kit (Invitrogen), plasmids were extracted from the obtained colonies, Respectively.
2) As a result, ssDNA having 9 different sequences was obtained, and the nucleotide sequences of nine different ssDNAs that specifically bind to E. coli with high affinity are shown in Table 1.
Example 7 Analysis of Secondary Structure of ssDNA
The above nine ssDNAs were amplified using the free Mfold program (http://mfold.rna.albany.edu/?q=mfold; Zuker, M. Nucleic Acids Res. 2003, 31, 3406) The structure of the car was analyzed. For example, the secondary structure of each of ssDNA having the nucleotide sequences of SEQ ID NOS: 5, 7, and 9 is shown in FIGS.
<Example 8> Analysis of binding force and selectivity to E. coli
1) Among the above 9 ssDNAs, 3 kinds (ssDNA having the nucleotide sequences of SEQ ID NOS: 5, 7 and 9) which are predicted to have a high binding ability to E. coli were selected through secondary structure analysis and the binding force and selectivity Respectively.
2) Analysis of binding force and selectivity to Escherichia coli was carried out by mixing 100 μL of fluorescently labeled ssDNA (0, 10, 25, 50, 100, 250, 500 nM) at various concentrations with Escherichia coli suspension prepared in Example 1, Lt; / RTI > After the reaction, ssDNA not bound to the surface of E. coli was washed three times using PBS buffer, and the fluorescence intensity of ssDNA bound to the surface of E. coli was measured. The fluorescence intensities at respective ssDNA concentration conditions were plotted using a SigmaPlot program with a nonlinear regression method and a single site saturated ligand binding method to determine the dissociation constant, Respectively.
[Equation 1]
F = B max * C / (K d + C )
(Where F is the fluorescence intensity, B max is the maximum binding position, K d is the dissociation constant, and C is the concentration of ssDNA)
3) The affinity analysis results are shown in FIGS. 5 to 7, and the dissociation constants of the three kinds of ssDNA sequences showing high affinity to E. coli are shown in Table 2 below. As a result, it was confirmed that ssDNAs having the nucleotide sequences of SEQ ID NOS: 5, 7, and 9 had strong affinity for E. coli.
≪ Example 9 >
1) Escherichia coli and Bacillus ( Bacillus spp.) Were used to confirm the selectivity of Escherichia coli against the three ssDNAs. subtilis , Klebsiella pneumoniae , Klebsiella pneumoniae ), Enterobacter aerogenes , and Staphylococcus epidermidis were used for selectivity analysis.
2) Experiments were performed by reacting 100 μL (10 7 cells) of each Escherichia coli and bacteria with 100 μL of 500 nM ssDNA for 1 hour at room temperature. After washing with PBS buffer, unbound ssDNA was removed and E. coli and bacteria The fluorescence intensity of the bound ssDNA was measured and compared.
3). The results are shown in FIG. 4. The ssDNA having the nucleotide sequences of SEQ ID NOS: 5, 7 and 9 exhibited fluorescence intensities of bacillus, clappsiella, citrobacter, enterobacter and staphylococcus Was found to be very low.
While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Should be interpreted as belonging to the scope.
<110> Korea Institute of Science and Technology <120> Screening method of DNA aptamer <130> PDAHJ-14155 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 1 gattagctac atttggttgt ttaccgctct gctttctatt attt 44 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 2 tgttagtgtt taaggcccaa agtcggttca tcagtacatt cctcg 45 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 3 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aag 43 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 4 gattgtggtg gggcctcgag atacctgcga ccggcatact tgaat 45 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 5 atttcgcccc cgtgttccga ctggtatctt cacgtcttcg agtgt 45 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 6 ttgcccgtac actgtcatcc tcggcttata gccattattg aaatt 45 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 7 cgcaatacca aagtggcgag agcgctgtct tgagtgagtg gttgg 45 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 8 gcgagggcca acggtggtta cgtcgctacg gcgctactgg ttgat 45 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli binding DNA aptamer <400> 9 tatggcgtgg caagcttggc ccgcttctca agcatggtta tctac 45 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Preserved region of ssDNA library <400> 10 tatggcgtgg caagcttggc ccgcttctca agcatggtta tctac 45 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Preserved region of ssDNA library <400> 11 caaaagtgca cgctactttg ctaa 24
Claims (11)
The reverse-discriminating substance is a substance different from the target substance among the substances capable of binding to the nucleic acid, and the re-collection step is repeated 9 to 39 times each, and the reverse-discriminating substance reaction step and the nucleic acid- Wherein the DNA amplification step is repeated 3 to 10 times, and the amplification step is performed only once.
Wherein the ssDNA library has a fixed primer region for PCR at both ends and has 30 to 50 random bases in the center.
Wherein the RPM in the centrifugation method is 10 to 100,000.
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| KR102243449B1 (en) * | 2019-10-07 | 2021-04-22 | 한국과학기술연구원 | A nucleic acid aptamer specifically binding to Enterobacter sakazakii and the use thereof |
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| KR101258600B1 (en) | 2010-12-30 | 2013-05-28 | 주식회사 아이센스 | DNA aptamer binding to ampicillin with specificity |
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| KR101258600B1 (en) | 2010-12-30 | 2013-05-28 | 주식회사 아이센스 | DNA aptamer binding to ampicillin with specificity |
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| Anal. Biochem., Vol. 436, No. 1, pp. 22-28 (2013.01.26.)* |
| Nat. Protoc., Vol. 5, No. 6, pp. 1169-1185 (2010.06.03.)* |
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| KR20220143453A (en) | 2021-04-16 | 2022-10-25 | 재단법인 대구경북첨단의료산업진흥재단 | Method of aptamer screening through primer region masking and NGS analysis of each round DNA pool |
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