CN103114147A - Aptamer screening method of no-fixed point target substance - Google Patents
Aptamer screening method of no-fixed point target substance Download PDFInfo
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- CN103114147A CN103114147A CN2013100638446A CN201310063844A CN103114147A CN 103114147 A CN103114147 A CN 103114147A CN 2013100638446 A CN2013100638446 A CN 2013100638446A CN 201310063844 A CN201310063844 A CN 201310063844A CN 103114147 A CN103114147 A CN 103114147A
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Abstract
The invention discloses an aptamer screening method of a no-fixed point target substance in the technical field of nucleic acid aptamer screening in analytical chemistry. The method comprises the following steps of: screening in a systematic evolution of ligands by an exponential enrichment (SELEX) method by building a random oligonucleotides library, and an upstream primer, a downstream primer and a fixed oligonucleotides library primer for polymerase chain reaction (PCR) proliferation and utilizing an agarose particle fixed oligonucleotides library with a streptavidin mark to obtain a PCR product for clone sequencing; and obtaining aptamers bonded with the no-fixed point target substance with the special affinity after the sequencing is finished. The method is applied to screening of heavy metal cadmium ion aptamers, so that 13 aptamers which are bonded with the cadmium ion with the high specificity and high affinity are obtained; and therefore, the aptamer screening method is good in fixing effect, high in adaptability, low in cost and suitable for aptamer screening of the no-fixed point target substance.
Description
Technical field
What the present invention relates to is the method in a kind of analytical chemistry aptamer triage techniques field, be specifically related to a kind of agarose particle immobilized oligonucleotide library that utilizes marked by streptavidin, and pass through the method for the aptamers of SELEX technology screening fixed point free target material.
Background technology
SELEX (Systematic Evolution of Ligands by Exponential Enrichment, the i.e. external evolution of index concentration Fas lignand system) technology is a kind of new combinatorial chemistry technique that grows up early 1990s.Utilize this technology can screen specificity and the affine aptamer of target material height from random single stranded oligonucleotide storehouse.Its basic ideas are one of external chemosynthesis single stranded oligonucleotide storehouses, mix with the target material, form target material-nucleic acid complexes, the unconjugated nucleic acid of wash-out, separate the nucleic acid molecule of being combined with the target material, and carry out pcr amplification take this nucleic acid molecule as template, then the screening that enters lower whorl.By screening and the amplification that repeats, some are not combined with the target material or have the nucleic acid molecule of low-affinity, middle avidity to be washed away with the target material, and the nucleic acid molecule that strong avidity is arranged with the target material from very large with separating hangar, and purity is carried out and is increased with the SELEX process, occupies at last great majority (〉 90% left and right in storehouse).At first use this technology screening after the specific nucleic acid aptamers of specific adsorption phage T4DNA polysaccharase and organic dye molecule from Tuerk and Ellington etc., through the development of more than ten years, the SELEX technology has become a kind of important research means and instrument.
Committed step in the SELEX process is that unconjugated nucleic acid is separated with the nucleic acid that can be combined with the target material.Most researchers is selected the target material is fixed, when mix with the target material in the single stranded oligonucleotide storehouse, can be with after the nucleic acid molecule of target material combination be stayed on mounting medium together with the target material, wash away can not in conjunction with nucleic acid.By the method for fixed target material, people use the SELEX technology successfully to screen to include the aptamers of the materials such as organic dye, medicine, amino acid, cytokine, cofactor, aminoglycoside, amino analogue, Nucleotide and polypeptide.But using the prerequisite of the method is that the target material has point of fixity, the rare report of screening of therefore relevant fixed point free target material aptamers.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of aptamers screening method of fixed point free target material is proposed, by the immobilized oligonucleotide library, the loose nucleic acid of wash-out, add target material and the nucleic acid effect of fixing during the forward screening, the nucleic acid molecule that can be combined with the target material under wash-out is used for the next round screening after pcr amplification; Oppositely add the material with target physical property or similar during screening, wash-out is removed the nucleic acid molecule with target material non-specific binding.Through the too much forward and reverse screening of round, obtain can with the aptamer of target material high specific, strong avidity effect.Present method is applied to the screening of heavy metal cadmium ion aptamers, obtains the aptamers of 13 energy and cadmium ion high specific, strong avidity effect.Good fixing effect of the present invention, suitability is strong and cost is low, can be used for the aptamers screening of fixed point free target material.
The present invention is achieved by the following technical solutions, the present invention is by building random oligonucleotide library and being used for the upstream primer of pcr amplification and downstream primer and immobilized oligonucleotide library primer, and utilize the agarose particle immobilized oligonucleotide library of marked by streptavidin, finally obtain can be used for the PCR product of cloning and sequencing by the screening of SELEX method, obtain the aptamers of being combined with fixed point free target material pathoklisis after order-checking.
The sequence of described random oligonucleotide library is:
5’-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3’，
Wherein: two ends are respectively the fixed sequence program of the 19bp shown in Seq ID No.1 and Seq ID No.2, and middle 30 bases are stochastic sequence.
Because each base in 30 bases may form by 4 kinds, so random oligonucleotide library theoretical library capacity is 4
30, can therefrom filter out in theory the aptamers of any target material.
Described upstream primer, namely the P1 sequence, as shown in Seq ID No.1, be 5 '-ACCGACCGTGCTGGACTCT-3 ';
Described downstream primer, namely the P2 sequence, as shown in Seq ID No.3, be 5 '-CGCAACGCTCGCTCATACT-3 ';
Described immobilized oligonucleotide library primer, i.e. P3 sequence, for
5’-Biotin-CGCAACGCTCGCTCATACT-3’
Wherein: the P3 sequence is by the Biotin(vitamin H, has with Streptavidin and pretends very much firmly) and Seq ID No.3 shown in downstream primer sequence composition.
Described immobilized oligonucleotide library refers to: carry out anneal after containing random oligonucleotide library and the aqueous solution that contains immobilized oligonucleotide library primer, then just the mixed solution after annealing joins in the pillar of the agarose that the surface grafting avidin is housed, and by the biotin-avidin effect, the random oligonucleotide storehouse is fixed on the agarose particle.
Described mixing refers to: take mol ratio as 1:2～and 3 ratio mixes.
Described anneal refers to: sex change 60s under 94 ° of C environment; Then 60min anneals under 59 ° of C environment; Again at 25 ° of C environment downward-extension 5min, wherein: the cooling rate of annealing process and the process of extension is 0.1 ° of C/s.
Fully wash through damping fluid before the screening of SELEX method in described immobilized oligonucleotide library, centrifugal loose oligonucleotide and the P3 sequence of removing.
Described SELEX method refers to: carry out successively some forward SELEX screenings of taking turns and oppositely SELEX screening.
Described forward SELEX screening refers to: add fixed point free target material to the agarose particle that is fixed with the random oligonucleotide storehouse, centrifugal treating and collect centrifugate after hatching, template as asymmetric PCR, collect product and purifying after the amplification of PCR program, use the library as follow-up forward screening, make fixed point free target material concentration reduce.
The concentration of described fixed point free target material can be set with reference to the relevant regulations of Environmental Protection Agency (EPA) and the World Health Organization (WHO).
Described hatching refers to: fixed point free target material acts on for some time at a certain temperature with the agarose particle that is fixed with the random oligonucleotide storehouse.
Described amplification refers to: enlarge in the centrifugate of above-mentioned collection can with the content of the oligonucleotide of target material effect.
The number of times of described forward SELEX screening is preferably and is reduced to standard detection limit (concrete concentration can with reference to the relevant regulations of Environmental Protection Agency (EPA) and the World Health Organization (WHO)) with fixed point free target material concentration and stops when following, and carries out reverse SELEX and screen.
Described reverse SELEX screening refers to: when the target material is certain heavy metal ion, first add different heavy metal ion from fixed point free target physical property or similar to the agarose particle that is fixed with the random oligonucleotide storehouse, hatch rear centrifugal treating and remove centrifugate, then add fixed point free target material, hatch rear centrifugal treating and collect centrifugate, as the template of pcr amplification oligonucleotide, collect product and purifying after the amplification of PCR program, as the library of follow-up reverse screening.
The number of times of described reverse SELEX screening is preferably 3～5 times.
principle of the present invention is: one of external chemosynthesis single stranded oligonucleotide storehouse, by the effect between vitamin H and avidin, single stranded oligonucleotide is fixed on medium, the loose Nucleotide of wash-out, during the forward screening, add fixed point free target material and fixing Nucleotide to hatch, hatch Nucleotide and the effect of target material generation affinity are arranged in process, this effect is enough to overcome the reactive force between vitamin H and avidin, when separating target material and mounting medium, the Nucleotide of bound fraction is just along with the target material breaks away from mounting medium, separate again the nucleic acid molecule of being combined with the target material, and carry out pcr amplification take this nucleic acid molecule as template, enter again the forward screening of lower whorl.By the forward screening and amplification that repeat, some are not combined with the target material or have the nucleic acid molecule of low-affinity, middle avidity to be left away with the target material.When oppositely screening, before adding the target material, first add and the target structure of matter or kin other materials and the nucleic acid effect of fixing, if there is nucleic acid molecule to break away from from mounting medium with this material, it is the non-specific binding nucleic acid of target material, also ought be removed.So through the too much forward and reverse screening of round, the nucleic acid molecule that high specific, strong avidity is arranged with the target material the most at last is from separating hangar, and this nucleic acid molecule is the aptamers of target material.
Technique effect
Compared with prior art, screening method provided by the invention does not need large-scale instrument and equipment, and good fixing effect, easy to operate, suitability is strong and cost is low, can be used for the aptamers screening of fixed point free target material.
Description of drawings
Fig. 1 is the technological line schematic diagram of cadmium ion aptamer screening of the present invention.
Embodiment
The below elaborates to embodiments of the invention, and the present embodiment is implemented under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
As shown in Figure 1, the present embodiment comprises the following steps:
Step 1) building total length is the random oligonucleotide library of 68bp, the sequence of oligonucleotide library is: 5 '-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3 ', wherein two ends are the fixed sequence program of 19bp, and middle 30 bases are stochastic sequence.
Step 2) three primers of design, wherein P1 (5 '-ACCGACCGTGCTGGACTCT-3 ') and P2 (5 '-CGCAACGCTCGCTCATACT-3 ') are used separately as upstream primer and the downstream primer of pcr amplification oligonucleotide, and P3 (5 '-Biotin-CGCAACGCTCGCT CATACT-3 ') is used for oligonucleotide library is fixed on the agarose particle of marked by streptavidin.
Step 3) get random oligonucleotide library, evenly mix 1:2～3 by volume with the P3 sequence, mixed solution carries out anneal, and annealing conditions is: 94 ° of C sex change 60s, and 59 ° of C annealing 60min, 25 ° of C extend 5min, and cooling rate is 0.1 ° of C/s.In annealing process, 19 bases of oligonucleotide 3 ' end will form pairing with P3.Mixed solution after annealing is joined in the pillar of the agarose that the surface grafting avidin is housed, by the biotin-avidin effect, the random oligonucleotide storehouse is fixed on the agarose particle.Fully wash centrifugal loose oligonucleotide and the P3 sequence of removing with damping fluid.
Step 4) add in the above-mentioned pillar certain 200 μ M cadmium ions to carry out the forward screening, hatch after certain hour centrifugally, collect centrifugate.
Step 5) centrifugate of above-mentioned collection is used as the template of asymmetric PCR, after certain PCR program amplification, collects PCR product and purifying, as the library of next round screening.
Step 6) by step 3) and 4) do the next round screening, often carry out taking turns screening, the concentration of cadmium ions that adds in step (4) is reduced.
Step 7) when concentration of cadmium ions is reduced to 0.002 μ M, by step 3)-5) oppositely screen.Step 4) before adding cadmium ion in, the metal ion that first adds other, hatch after certain hour centrifugally, discard centrifugate, then add 200 μ M cadmium ions in pillar, hatch after certain hour centrifugal, collect centrifugate, as the template of pcr amplification oligonucleotide, after certain PCR program amplification, collect PCR product and purifying, as the library of the reverse screening of next round.Take turns through ten the interpretation of result that obtains after forward SELEX screening as follows:
Step 8) carry out four-wheel oppositely after screening, the PCR product that gained is reclaimed purifying is transferred to professional DNA sequencing company and is carried out cloning and sequencing.
Step 9) cloning and sequencing obtains 13 cadmium ion aptamer sequences.
Claims (10)
1. the aptamers screening method of a fixed point free target material, it is characterized in that, by building random oligonucleotide library and being used for the upstream primer of pcr amplification and downstream primer and immobilized oligonucleotide library primer, and utilize the agarose particle immobilized oligonucleotide library of marked by streptavidin, finally obtain can be used for the PCR product of cloning and sequencing by the screening of SELEX method, obtain the aptamers of being combined with fixed point free target material pathoklisis after order-checking;
The sequence of described random oligonucleotide library is:
5 '-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3 ', wherein: two ends are respectively the fixed sequence program of the 19bp shown in Seq ID No.1 and Seq ID No.2, and middle 30 bases are stochastic sequence;
Described upstream primer, i.e. P1 sequence is as shown in Seq ID No.1;
Described downstream primer, i.e. P2 sequence is as shown in Seq ID No.3;
Described immobilized oligonucleotide library primer, i.e. P3 sequence, be 5 '-Biotin-CGCAACGCTCGCTCATACT-3 ' wherein: the P3 sequence is comprised of the downstream primer sequence shown in Biotin and Seq ID No.3.
2. method according to claim 1, it is characterized in that, described immobilized oligonucleotide library refers to: carry out anneal after containing random oligonucleotide library and the aqueous solution that contains immobilized oligonucleotide library primer, then just the mixed solution after annealing joins in the pillar of the agarose that the surface grafting avidin is housed, and by the biotin-avidin effect, the random oligonucleotide storehouse is fixed on the agarose particle.
3. method according to claim 2, is characterized in that, described mixing refers to: take mol ratio as 1:2～and 3 ratio mixes.
4. method according to claim 2, is characterized in that, described anneal refers to: sex change 60s under 94 ° of C environment; Then 60min anneals under 59 ° of C environment; Again at 25 ° of C environment downward-extension 5min, wherein: the cooling rate of annealing process and the process of extension is 0.1 ° of C/s.
5. method according to claim 1 and 2, is characterized in that, fully wash through damping fluid before the screening of SELEX method in described immobilized oligonucleotide library, centrifugal loose oligonucleotide and the P3 sequence of removing.
6. method according to claim 1, is characterized in that, described SELEX method refers to: carry out successively some forward SELEX screenings of taking turns and oppositely SELEX screening.
7. method according to claim 6, it is characterized in that, described forward SELEX screening refers to: add fixed point free target material to the agarose particle that is fixed with the random oligonucleotide storehouse, centrifugal treating and collect centrifugate after hatching, template as asymmetric PCR, collect product and purifying after the amplification of PCR program, use the library as follow-up forward screening, make fixed point free target material concentration reduce.
8. according to claim 6 or 7 described methods, is characterized in that, the number of times of described forward SELEX screening stops when following for be reduced to the standard detection limit with fixed point free target material concentration, and carries out reverse SELEX screening.
9. method according to claim 6, it is characterized in that, described reverse SELEX screening refers to: when the target material is certain heavy metal ion, first add different heavy metal ion from fixed point free target physical property or similar to the agarose particle that is fixed with the random oligonucleotide storehouse, hatch rear centrifugal treating and remove centrifugate, then add fixed point free target material, hatch rear centrifugal treating and collect centrifugate, template as the pcr amplification oligonucleotide, collect product and purifying after the amplification of PCR program, as the library of follow-up reverse screening.
10. according to claim 6 or 9 described methods, is characterized in that, the number of times of described reverse SELEX screening is 3～5 times.
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| CN104862317A (en) * | 2015-05-18 | 2015-08-26 | 江汉大学 | Hepatoma cell-specific aptamer and preparation method thereof |
| CN104634915B (en) * | 2013-11-08 | 2017-03-29 | 中国科学院大连化学物理研究所 | A kind of granule of oligonucleotide library modification and its preparation and application |
| CN109321564A (en) * | 2018-10-30 | 2019-02-12 | 廖世奇 | A kind of fusion protein aptamer screening technique and kit |
| WO2019154410A1 (en) * | 2018-02-12 | 2019-08-15 | 翼高生物科技有限公司 | Aptamer for sclerostin and use thereof |
| CN113652432A (en) * | 2021-08-20 | 2021-11-16 | 山东理工大学 | Aminoglycoside antibiotic broad-spectrum aptamer |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104634915B (en) * | 2013-11-08 | 2017-03-29 | 中国科学院大连化学物理研究所 | A kind of granule of oligonucleotide library modification and its preparation and application |
| CN104862317A (en) * | 2015-05-18 | 2015-08-26 | 江汉大学 | Hepatoma cell-specific aptamer and preparation method thereof |
| WO2019154410A1 (en) * | 2018-02-12 | 2019-08-15 | 翼高生物科技有限公司 | Aptamer for sclerostin and use thereof |
| CN109321564A (en) * | 2018-10-30 | 2019-02-12 | 廖世奇 | A kind of fusion protein aptamer screening technique and kit |
| CN113652432A (en) * | 2021-08-20 | 2021-11-16 | 山东理工大学 | Aminoglycoside antibiotic broad-spectrum aptamer |
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Inventor after: Zhou Pei Inventor after: Wu Yuangen Inventor after: Zhan Shenshan Inventor after: Wang Faze Inventor after: Zhong Lingying Inventor after: Liu Le Inventor after: Xing Haibo Inventor after: Zhan Xuejia Inventor before: Zhou Pei Inventor before: Wu Yuangen Inventor before: Zhan Shenshan Inventor before: Wang Faze Inventor before: Liu Le Inventor before: Xing Haibo Inventor before: Zhan Xuejia |
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