JP2008237041A - Method for selecting target cell specifically binding nucleic acid by annealing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily obtaining a DNA that specifically binds target cells, but not non-target cells. <P>SOLUTION: The target cell specifically binding DNA is obtained by selecting target cell specifically binding DNA candidates, selecting antisense strand DNAs of non-target cell specifically binding DNAs from the candidates and deleting the non-target cell specifically binding DNAs from the target cell specifically binding DNA candidates. Target cell specifically binding DNAs are determined for a plurality of target cells, the target cell specifically binding DNAs are contrasted for the two target cells and the coexisting DNAs are made DNAs specifically binding the final target cell. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for selecting a nucleic acid that specifically binds to a target cell. In particular, the present invention relates to a selection method by annealing nucleic acids that specifically bind to target cells.

  It was reported in 1990 by a group of C. Tuerk et al. That non-patent literature 1: Science, 249, 505-510 ( 1990)) and these nucleic acids were named “aptamers”. Previous studies have reported many aptamers that have affinity for ions, sugar chains, proteins, molecules on the surface of living cells, and aptamers that determine the phosphorylation state of proteins. Aptamers are roughly divided into single-stranded DNA or single-stranded RNA, but other peptide aptamers have been reported. Among them, modified aptamers according to the purpose of use, such as aptamers using artificial bases and aptamers modified with organic molecules such as polyethylene glycol, have been developed.

  These aptamers are selected by the two steps of SELEX (Systematic Evolution of Ligands by Exponential Enrichment) proposed by C. Tuerk et al., Selection of target nucleic acid molecules and amplification of those molecules.

In general, first, a nucleic acid pool having a total length of about 60 to 140 bases consisting of a random sequence of 30 to 100 bases sandwiched between primer sequences of 10 ¹⁴ to 10 ¹⁶ molecular species and ¹⁸ to 38 bases at both ends is prepared. prepare. Next, a sequence having an affinity for the target molecule is selected from the nucleic acid pool. Various methods have been developed for this operation. In general, when nucleic acids having affinity are selected using only target molecules, aptamers that have affinity for non-target molecules ("cross reactivity" in antibodies) are also included in the final obtained aptamers. It is not unusual to exist. For this reason, aptamers that have affinity for target molecules but no affinity for non-target molecules in samples in which many types of molecules that are expected to be used are mixed can be selected by the counter selection method (Counter Selection). ).

Here, as an example, the following target cells reported by Dihua Shangguan et al. In 2006 (Non-Patent Document 2: Proceedings of the National Academy of Sciences of the United States of America, 103, 11838-11843) A method for selecting a DNA aptamer that specifically binds will be briefly described. Dihua Shangguan et al. Used a T cell line (CCRF-CEM cell) and a B cell line (Ramos cell), and has a single-stranded DNA aptamer that has affinity for the former but no affinity for the latter. Were sorted by the counter sorting method. The CCRF-CEM cell used here is a cell line of human acute lymphoblastic leukemia / T cell and is widely used as a model cell for human leukemia. First, a single-stranded DNA pool was prepared as a nucleic acid pool to be used. Next, this single-stranded DNA pool was applied to a T cell line (1-2 × 10 ⁶ cells) as a target cell. Thereafter, the cells were precipitated by centrifugation, unbound single-stranded DNA contained in the supernatant was removed, and single-stranded DNA bound to the cells was recovered. The collected single-stranded DNA was applied to a B cell line which is a non-target cell, and the cells were precipitated by centrifugation. On the contrary, unbound single-stranded DNA contained in the supernatant was collected. The collected single-stranded DNA was amplified by PCR. After 20 cycles of this process, cloning was performed in E. coli by the TA cloning method, and the sequence of the finally obtained single-stranded DNA was determined.

By the above operation, it is reported that a single-stranded DNA aptamer having affinity for the T cell line but not affinity for the B cell line was obtained. Furthermore, in the T cell line (CCRF-CEM cell) population that was considered to be a population of cells with uniform properties, it was reported that single-stranded DNA aptamers having affinity only for some of the populations were also obtained. is doing. In addition, among them, an aptamer having an affinity only for CCRF-CEM cells was present in a sample obtained by adding the CCRF-CEM cells to blood collected from normal human bone marrow in which a plurality of types of cells were actually mixed. Reporting. Furthermore, it has been reported that aptamers that have no affinity for B cells collected from the bone marrow of B lymphoma patients and have affinity only for T cells collected from the bone marrow of T lymphoma patients existed. It shows that there is sufficient potential as a basic technology aimed at application.
Science, 249, 505-510 (1990) Proceedings of the National Academy of Sciences of the United States of America, 103, 11838-11843

  Thus, in the conventional SELEX method, a nucleic acid pool is bound to a target molecule and recovered, and after amplification by PCR (after being applied to a non-target cell if the operation of the counter-selection method is performed), the nucleic acid is re-targeted. Perform the operation of binding to. The above example was for DNA, but in the case of RNA, the RNA bound to the target cell was recovered and (again, after being applied to the non-target cell, if subjected to the counter sorting method) RT- After amplification by PCR, the RNA pool is regenerated by in vitro transcription and again bound to the target molecule. Normally, aptamers cannot be selected only by performing this process once, and therefore it is necessary to repeat 10 to 20 times. The counter selection method for removing nucleic acids having affinity for non-target molecules must also be performed each round, so that the operation is very complicated and requires time.

  This is because, in the initial stage of selection, the absolute amount and the relative amount of the nucleic acid to be selected are small, and it is difficult to separate only the nucleic acid to be selected from this. If a method capable of removing only sequences other than those scheduled to be selected is developed, the operation will be very simple. In the above example, one type is used as a non-target cell, but a plurality of types of non-target cells are used to select nucleic acids that have no affinity for all of them but only have an affinity for the target cell. No report has been made. However, as in the above example, it has been reported that the finally selected nucleic acid has no affinity for all of the plural types of non-target cells but has an affinity only for the target cells.

  Further, as in the above example, in the conventional SELEX method, in a cell population that is considered to be a population of cells having uniform properties, a nucleic acid having affinity only for a certain cell population may be obtained. is there. In this way, even with a population of cells with uniform properties, in fact, there are limitations on the classification of cell populations in studies using conventional antibodies, and aptamers that can recognize the properties of target cells in more detail. This report suggests that more detailed classification is possible by using it.

  Furthermore, it is expected that differences in the properties of individual cells can be detected by performing this classification on individual cells. In conventional research, for example, the results obtained in cell membrane protein expression analysis, etc. are the results obtained with a cell population on the premise that they handle a population of cells with uniform properties. It's like an average of the values you have. Therefore, if SELEX is performed at the level of one cell and a nucleic acid having affinity for each cell constituting the cell population can be obtained, it is expected that the properties of each cell in the cell population can be determined in more detail. The

  Therefore, an object of the present invention is to propose a method capable of selecting a nucleic acid that specifically binds to a target cell in a short time and does not bind to a non-target cell, and also proposes a method capable of selecting it at the single cell level. It is to be.

Therefore, the present invention provides the following nucleic acid selection method and nucleic acid production method.
(1) (i) separating the target cell group and the non-target cell group from the cell group;
(Ii) applying a single-stranded DNA pool to target cells to bind single-stranded DNA that specifically binds to the target cells;
(Iii) a target cell-specific candidate DNA pool comprising target cell-specific candidate DNAs that are selected from the target cells bound to the specifically-bound single-stranded DNA and bound to the selected target cells The process of creating,
(Iv) applying the target cell-specific candidate DNA pool to non-target cells to bind the target cell-specific candidate DNA that specifically binds to the non-target cells to the non-target cells;
(V) creating a non-target cell-specific DNA pool comprising target cell-specific candidate DNAs bound to the non-target cells to which target cell-specific candidate DNAs that specifically bind to the non-target cells are bound And (vi) removing from the target cell-specific candidate DNA obtained in step (iii) the DNA complementary to the non-target cell-specific DNA obtained in step (v) by annealing. Determining a target cell-specific DNA,
A method for selecting a nucleic acid that specifically binds to a target cell.
(2) further comprising the step of performing the steps (i) to (vi) on a plurality of different target cells,
A DNA having a nucleic acid sequence that matches with respect to all the target cell-specific DNA pools by contrasting each target cell-specific DNA pool containing the target cell-specific DNA determined for each of the different target cells The nucleic acid selection method according to (1) above, wherein is determined as a target cell-specific DNA for the target cell group.
(3) (i) separating the target cell group and the non-target cell group from the cell group;
(Ii) applying a single-stranded DNA pool to target cells to bind single-stranded DNA that specifically binds to the target cells;
(Iii) The target cell to which the specifically bound single-stranded DNA is bound is selected, and the single-stranded DNA bound to the selected target cell is used as the sense strand DNA and bound. Amplifying single-stranded DNA by one-cell real-time PCR;
(Iv) selecting the sense strand DNA of the amplified single-stranded DNA to create a target cell-specific candidate sense strand DNA pool;
(V) Applying the target cell-specific candidate sense strand DNA pool to the non-target cells to bind the target cell-specific candidate sense strand DNA that specifically binds to the non-target cells to the non-target cells Process,
(Vi) the target using the target cell-specific candidate sense strand DNA bound to the non-target cell to which the target cell-specific candidate sense strand DNA bound specifically to the non-target cell is bound as the sense strand DNA; Amplifying the cell-specific candidate sense strand DNA by PCR;
(Vii) selecting the antisense strand DNA of the amplified target cell-specific candidate sense strand DNA to create a non-target cell-specific antisense strand DNA pool; and (viii) obtained in step (iv) Removing from the target cell-specific candidate sense strand DNA, the DNA complementary to the non-target cell-specific antisense strand DNA obtained in step (vii) by annealing,
A method for selecting a nucleic acid that specifically binds to a target cell.
(4) The above step (i)
Applying the magnetic beads on which the antibody that binds to the target cells is immobilized to the cell group to magnetically separate the target cells to which the magnetic beads are bound;
The nucleic acid selection method according to (3) above, comprising:
(5) The above step (vi)
Amplifying the target cell-specific candidate sense strand DNA by real-time PCR;
The nucleic acid selection method according to (3) above, comprising:
(6) In the above steps (iii) and (iv),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is removed by a filter, and only the single strand DNA sense strand DNA is selected to create a target cell-specific candidate sense strand DNA pool.
The nucleic acid selection method according to (3) above.
(7) In the above steps (iii) and (iv),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is magnetically removed using streptavidin magnetic beads that bind to biotin, and only the single strand DNA sense strand DNA is selected, and the target cell specific candidate sense strand DNA is selected. A pool is created,
The nucleic acid selection method according to (3) above.
(8) In the above steps (iii) and (vi),
Primer for amplification of sense strand DNA is modified with biotin, primer for amplification of antisense strand DNA is modified with amino group,
The amplified sense strand DNA is removed by a filter to create an antisense strand DNA pool of non-target cell specific candidate sense strand DNA pools.
The nucleic acid selection method according to (3) above.
(9) In the above steps (iii) and (vi),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is left magnetically using streptavidin magnetic beads that bind to biotin, and the non-target cell-specific candidate sense strand DNA is removed, and the non-target cell-specific candidate sense strand is removed. An antisense strand DNA pool of the DNA pool is created;
The nucleic acid selection method according to (3) above.
(10) The step (viii)
Fixing the antisense strand DNA of the non-target cell-specific DNA to a carrier provided in a chromatography column, and introducing the target cell-specific candidate sense strand DNA into the chromatography column; Removing the target cell-specific candidate sense strand DNA complementary to the antisense strand DNA of the non-target cell-specific DNA by annealing,
The nucleic acid selection method according to (8) above, comprising:
(11) The nucleic acid selection method according to (10), which comprises amplifying the target cell-specific candidate sense strand DNA flowing out from the chromatography column by real-time PCR and repeatedly introducing the DNA into the chromatography column. .
(12) The above step (viii)
The target cell-specific candidate sense strand DNA is introduced into the magnetically left antisense strand DNA using the streptavidin magnetic beads that bind to the biotin, and the non-target cell-specific DNA antisense strand DNA and Removing the complementary target cell-specific candidate sense strand DNA by annealing,
The nucleic acid selection method according to (9) above, comprising:
(13) The target cell-specific candidate sense strand DNA remaining after removal of the target cell-specific candidate sense strand DNA complementary to the antisense strand DNA of the non-target cell-specific sense strand DNA by annealing is subjected to real-time PCR. The target cell-specific candidate sense strand DNA is repeatedly introduced into the magnetically left antisense strand DNA using the streptavidin magnetic beads that are amplified and bound to the biotin, and the non-target cell-specific DNA The method for selecting a nucleic acid according to (12) above, comprising removing the target cell-specific candidate sense strand DNA complementary to the antisense strand DNA by annealing.
(14) The method further includes the step of performing the steps (i) to (viii) on a plurality of different target cells,
A DNA having a nucleic acid sequence that matches with respect to all the target cell-specific DNA pools by contrasting each target cell-specific DNA pool containing the target cell-specific DNA determined for each of the different target cells The nucleic acid selection method according to (3), wherein is determined as a target cell-specific DNA for the target cell group.
(15) A method for producing a nucleic acid that specifically binds to a target cell, comprising a step of using the nucleic acid selection method according to any one of (1) to (14) above.

In the present invention, a nucleic acid having affinity only for target cells and not affinity for non-target cells (a plurality of non-target cells may be selected) is selected. This selection process is as follows.
First, target cells are selected from the cell group. Next, the nucleic acid pool is applied to the target cells, and nucleic acids having affinity for the target cells are selected. At this stage, there are also nucleic acids that have affinity for non-target cells.
Therefore, next, the nucleic acid pool having affinity for the selected target cells is also applied to non-target cells to separate nucleic acids having affinity for non-target cells.
Next, by separating and removing the nucleic acid having affinity for the non-target cell from the nucleic acid having affinity for the target cell, not only specifically binding to the target cell but also affinity for the non-target cell. Only nucleic acids that do not have are selected.
In addition to the above-described selection in the first stage, the target cells are further compared with the nucleic acids having an affinity for each target cell, and only the nucleic acids present in all the individual target cells compared are selected (second stage). By performing the above, it is possible to obtain a nucleic acid that specifically binds to a stricter target cell.

  According to the present invention, a nucleic acid not only specifically binding to a target cell but also having no affinity for a non-target cell can be easily obtained in a short time, and further a nucleic acid at a single cell level that is not a cell population. Can be obtained.

  FIG. 1 is a flowchart for explaining a first stage for carrying out the present invention. That is, it is a stage that not only specifically binds to target cells but also selects only nucleic acids that have no affinity for non-target cells.

FIG. 1 (A) is a process of separating a cell group including a large number of cells composed of target cells and non-target cells into a cell group including target cells and a cell group including non-target cells. Shows the state of separation into target cells A ₁ , A ₂ , A ₃ , ---, _Ak and non-target cells B, C, D, ---, Z using antibodies that specifically bind to . Here, the target cells _{A 1, A 2, A 3} , ---, nucleic acids were indicated as A _k can be the same target cells, which present some of the difference in nature, that specifically bind to these This is to explain that the selection can be performed in consideration of these.

FIG. 1 (B) is a diagram showing the application of a single-stranded DNA pool of the 10 ¹⁵ level type prepared in advance to the separated target cells.

Figure 1 (C) is a diagram schematically showing a state where the nucleic acid is bound to a target cell _{A 1, A 2, A 3} , A k respectively. The target cell A ₁ has nucleic acids A, B, a, b, d, the target cell A ₂ has nucleic acids A, B, a, c, e, and the target cell A ₃ has nucleic acids A, C, b, f. , G and nucleic acid A, D, b, e, f are bound to the target cell _Ak , respectively. Therefore, regarding the target cell A ₁ , the target cell-specific candidate DNA pools are nucleic acids A, B, a, b, and d. Similarly, for the target cell A ₂ , the target cell-specific candidate DNA pools are nucleic acids A, B, a, c, and e.

  FIG. 1 (D) is a diagram showing that nucleic acid A, B, a, b, d, which is a target cell-specific candidate DNA pool, is applied to non-target cells B, C, D, ---, Z. .

FIG. 1 (E) is a diagram schematically showing a state in which a nucleic acid is bound to each of non-target cells B, C, D, and E. Nucleic acids B and d bind to non-target cell B, nucleic acids B and d bind to non-target cell C, nucleic acid b binds to non-target cell D, and nucleic acids B and d bind to non-target cell E, respectively. ing. Therefore, nucleic acids B, b, and d are obtained as non-target cell-specific DNA pools from all the nucleic acids bound to these non-target cells. As a result, it is found that among the nucleic acids A, B, a, b, and d of the target cell-specific candidate DNA pool for the target cell A ₁ , the nucleic acids B, b, and d are inappropriate. .

  FIG. 1F is a diagram showing this processing. That is, the nucleic acid B, b, d of the non-target cell specific DNA pool is removed from the nucleic acid A, B, a, b, d of the target cell specific candidate DNA pool, and the nucleic acid A, a is the target cell specific DNA pool. Indicates that there is. In this process, for example, as shown in the lower part of FIG. 2A, the non-target cell-specific DNA pool nucleic acids B, b, and d are immobilized on a carrier NHS 42 provided in a chromatography column 41. It can be easily carried out by removing the nucleic acid trapped in this.

In this way, a nucleic acid that specifically binds specifically to the target cell can be obtained by the first stage treatment. However, as can be easily seen from FIG. 1 (C) and FIG. 1 (F), for example, the target cell-specific candidate DNA pool nucleic acid A, B, a, c, e for the target cell A _{2 is} non-target cell specific. If the nucleic acid B, b, d in the target DNA pool is removed, the nucleic acid in the target cell specific DNA pool becomes A, a, c, e. This may be accomplished, the nucleic acid of the target cell-specific DNA pool of target cells A is during the processing of FIG. 1 (F), which of the target cells A _1, A _2, A _3, A _k is determined is selected This means that the selection of nucleic acids in the target cell-specific DNA pool of target cell A cannot be uniquely determined.

  In the second stage, paying attention to this, processing for selecting nucleic acids in the target cell-specific DNA pool more precisely is performed. The second stage will be described with reference to FIG.

FIG. 2 (A) shows a process of removing nucleic acids B, b, and d of non-target cell-specific DNA pools from target cell-specific candidate DNA pools of target cells A ₁ , A ₂ , A ₃ , and _Ak , respectively. The nucleic acid of the target cell specific DNA pool of the result was shown. Thus, it can be seen that target cell-specific DNA in a strict sense cannot be determined only by the first stage.

FIG. 2 (B) shows that nucleic acid A, a which is a target cell-specific DNA pool of target cell A ₁ from target cell-specific DNA pools of target cells A ₁ , A ₂ , A ₃ and _Ak. The target cell-specific DNA pool nucleic acid A, a obtained by ANDing the target cell-specific DNA pool nucleic acid A, a, c, e of the target cell A ₂ is obtained. Then, the nucleic acid A target cell-specific DNA pool to which this AND is taken, a target cells A ₃ of target cell-specific DNA pool of nucleic acids A, C, g, the target cell-specific DNA pools 0.00 AND of f To obtain nucleic acid A. Then, the nucleic acid A and a target cell A _k The AND is a target cell-specific DNA pools get the target cell-specific DNA pools nucleic A, D, g, the target cell specific DNA pools 0.00 AND of f Nucleic acid A is obtained. As described above, the target cell-specific DNA pools of the target cells A ₁ , A ₂ , A ₃ , and A _k are made to correspond to each other and narrowed down to those that satisfy the AND condition step by step. Determine the target cell specific DNA in meaning. For example, as shown in the lower part of FIG. 2B, this treatment is performed by, for example, supporting the nucleic acid A, a of the target cell-specific DNA pool of the target cell A _{1 in} the chromatography column 41. The nucleic acid A, a, c, e of the target cell-specific DNA pool of the target cell A ₂ is applied to the NHS 42 and the nucleic acid trapped on the carrier NHS 42 is obtained. The nucleic acid trapped on the carrier NHS42 is taken out. Next, the extracted nucleic acid is immobilized on a carrier NHS 42 provided in the chromatography column 41, and the nucleic acid A, C, g, f of the target cell-specific DNA pool of the target cell A ₃ is applied thereto. Nucleic acids trapped on the carrier NHS42 are obtained. The nucleic acid trapped on the carrier NHS42 is taken out. It can be easily executed by repeating the procedure.

  In each of the processes described above, it is natural to amplify the nucleic acid by PCR if necessary. Further, the operation for obtaining the nucleic acid of the target cell-specific DNA pool of the target cell described above is not limited to one cell, but a small number of cells are treated as a group, and this is performed on the target cell-specific DNA of the target cell of the above-mentioned one cell It is good as a pool. Specific processing will be described below.

Example 1
In Example 1, a nucleic acid that specifically binds to a target cell is obtained by the following procedure. First, the first stage process will be described.
(I) Separation of target and non-target cells

(1) Prepare magnetic beads on which an antibody that binds to the surface of a target cell is immobilized, and mix with a buffer in which a cell group is suspended. FIG. 3 (A) is a diagram schematically showing this state, in which 11 ₁ is a test tube, 12 ₁ is a buffer, 13 is a target cell, 14 is a non-target cell, and 15 is a magnetic bead on which an antibody is immobilized. is there. After waiting for the magnetic beads 15 with the antibody immobilized in the buffer 12 ₁ to bind to the target cells 13, the magnet 16 is brought close to the outside of the test tube 11 ₁ to attract the magnetic beads 15. In this state, the non-target cells 14 to which the magnetic beads 15 are not bound together with the buffer 12 ₁ are moved to another test tube 11 ₂ to perform magnetic separation of the cells. Figure 3 (B), (C) is a diagram showing this state schematically, target cell 13 in which the magnetic beads 15 antibody is immobilized bound remains in the test tube 11 _1, magnetic beads 15 are bonded non-target cells 14 were not transferred to the test tube 11 _2. The method of magnetic separation is performed according to established procedures.

(2) In order to select a nucleic acid candidate that can be a nucleic acid that specifically binds to the target cell of the present invention, a fixed part consisting of a predetermined number of unique sequence bases on the 5 ′ end side, 3 ′ end A single-stranded DNA pool consisting of a random part having a fixed part consisting of a predetermined number of unique sequence bases and having a random base sequence between the two fixed parts is prepared. The base sequences of the 5 ′ terminal side fixing part and the 3 ′ terminal side fixing part may be the same, the number of bases may be 18 to 38, and the number of bases in the random part may be about 30 to 100. The single-stranded DNA pool may be about 10 ¹⁵ kinds of DNA as a whole. This single-stranded DNA pool may be created with a nucleic acid synthesizer, or a commercially available one may be purchased. This is schematically shown in the upper part of FIG. 21 ₁ , 21 ₂ , ---, and 21 _n are reference numbers that mean DNA, respectively, the filled portions shown on both sides are fixed portions of the base sequence, and the pattern notation sandwiched between these fixed portions is the random number of the base sequence Part.

The DNA of the single-stranded DNA pool is heat-denatured at 95 ° C. and then rapidly cooled on ice to make the higher-order structure of each DNA in the single-stranded DNA pool uniform. The DNA maintaining the higher order structure is added to the buffer 12 ₂ of the test tube 11 _{1 where} the target cells 13 are collected, and allowed to bind to the cells on ice. At this time, DNA of several sequences out of about 10 ¹⁵ kinds of DNA binds to the target cell 13. Actually, DNA having a higher affinity binds more. This is schematically shown on the left side of the lower part of FIG. FIG. 4 schematically shows a state in which four of the seven DNA patterns schematically shown in the single-stranded DNA pool are bound to the target cell 13. At this time, the DNA bound to the target cell 13 may be a DNA having a sequence that specifically binds to the target cell 13. As the buffer 12 ₂ used at this time, a standard buffer such as a buffer containing magnesium ion Mg ²⁺ is used. Further, at the time of binding, yeast tRNA and bovine serum albumin (BSA) are added to suppress non-specific binding. Is good.
(II) Amplification of DNA binding to target cells

(3) Next, any one cell to which the single-stranded DNA is bound is taken out of the test tube 11 ₁ .

(4) Next, single-stranded DNA bound on the cell surface is amplified by single-cell real-time PCR. Here, it is assumed that DNA 21 ₁ of single-stranded DNA pool is amplified. At this time, for amplification of the sense strand DNA, the primer 23 complementary to the fixed part at the 5 ′ end is used without modification. On the other hand, for amplification of antisense strand DNA, a primer 22 complementary to the fixed part at the 5 ′ end is modified with biotin 24 and used. This is schematically shown in the lower center of FIG. Here, in order to increase the S / N ratio between DNA that specifically binds to the target cell 13 and DNA that binds non-specifically, the amount of amplification is confirmed by real-time PCR, and the reaction is terminated before reaching the plateau. Is good. The conditions for real-time PCR follow an already established method.

(5) Next, after real-time PCR, unreacted primers are removed by electrophoresis, the double strands are dissociated by alkali treatment, and biotin 24 is modified at the 5 ′ end of unnecessary antisense strand DNA. It is removed using a filter using what is being done. This is schematically shown on the right side at the bottom of FIG. Buffer containing DNA after real-time PCR, are injected into the test tube 11 ₃ having a filter 26 which does not transmit biotin 24. Therefore, the buffer 27 that has passed through the filter 26 has only the sense strand DNA of DNA that may be DNA having a sequence that specifically binds to the target cell 13. In FIG. 4, this is indicated as a target cell-specific candidate DNA pool. In the example of the figure, four DNAs bound to the target cell 13 of the test tube 11 ₁ correspond.

(6) Next, the obtained target cell-specific candidate DNA pool is neutralized with dilute hydrochloric acid HCl or the like and then washed with a general reagent such as TE buffer. Thereby, the sense strand DNA denatured by alkali is recovered by alkali treatment for removing the antisense strand DNA. In FIG. 4, this process is not shown.
(III) Amplification of antisense strand DNA of DNA that binds to non-target cells 14

(7) Next, as shown in FIG. 3C, in the buffer 12 ₂ of the test tube 11 ₂ containing the non-target cells 14 to which the magnetic beads 15 on which the antibody is immobilized are not bound, The target cell-specific candidate DNA pool DNA obtained in the operation (6) is injected to bind to the non-target cells 14. This is schematically shown on the left side of FIG. FIG. 5 schematically shows a state in which three DNAs among the four DNA patterns schematically shown in the target cell-specific candidate DNA pool are bound to the non-target cells 14. At this time, the DNA bound to the non-target cell 14 is a DNA having a sequence that specifically binds to the target cell 13 and may also specifically bind to the non-target cell 14.

(8) Next, collect all cells in vitro 11 ₂ by centrifugal, amplifying the bound DNA on their cell surface by conventional PCR. Here, DNA21 _{2 of the} single-stranded DNA pool was amplified. At this time, a primer 23 in which biotin 24 is modified at the 5 ′ end is used for amplification of the sense strand DNA, and a primer in which the amino group 25 is modified at the 5 ′ end is used for amplification of the antisense strand DNA. This is schematically shown in the center of FIG. The PCR performed here is performed according to a conventional method that has already been established.

(9) Next, after PCR, unreacted primers are removed by electrophoresis, the double strands are dissociated by alkali treatment, and biotin 24 is modified at the 5 ′ end of unnecessary sense strand DNA. It removes using a filter using that. This is schematically shown on the right side of FIG. Buffer 12 ₂ containing DNA after PCR is injected into a test tube 35 having a filter 26 which does not transmit biotin 24. Therefore, the buffer 37 that has passed through the filter 26 has only the amino group-modified antisense strand DNA of DNA having a sequence that specifically binds to the non-target cells 14. In FIG. 5, this was indicated as a non-target cell-specific antisense strand DNA pool. In the example of the figure, three of the four DNAs bound to the target cell 13 of the test tube 11 ₁ correspond.

(10) Next, the antisense strand DNA of the obtained non-target cell-specific DNA pool is neutralized with dilute hydrochloric acid HCl or the like and then washed with a general reagent such as TE buffer. Thereby, the antisense strand DNA denatured by alkali is recovered by alkali treatment for removing the sense strand DNA. In FIG. 5, this process is not shown.
(IV) DNA purification by affinity chromatography

  (11) Next, the antisense strand DNA of the non-target cell-specific antisense strand DNA pool obtained in the process of (10) above is immobilized on a carrier NHS42 or the like provided in the chromatography column 41. This is schematically shown in the center of FIG. The immobilization of DNA on the carrier is carried out according to an established conventional method.

  (12) Next, the entire column 41 is brought to 95 ° C., and the DNA fixed to the carrier 42 is heat denatured.

  (13) Next, the DNA of the target cell-specific candidate sense strand DNA pool purified in the step (6) of amplification of DNA that binds to the target cell (6) is heat denatured at 95 ° C. and added to the column 41. . This is schematically shown in the upper part of FIG.

  (14) Next, by holding the column 41 at the annealing temperature, the target cell-specific candidate sense strand DNA is paired with the antisense strand DNA of the non-target cell-specific DNA, whereby complementary DNA is obtained. Trap in the column 41. As a result, the DNA complementary to the antisense strand DNA of the non-target cell-specific DNA in the target cell-specific candidate sense strand DNA is trapped in the column 41, and conversely, the antisense of the non-target cell-specific DNA is antisenseed. Only DNA that is not complementary to the strand DNA flows out of the column 41. This is expressed as target cell-specific sense strand DNA and is schematically shown at the bottom of FIG.

(15) Of course, the DNA flowing out from the column 41 includes not only sense strand DNA that specifically binds to the target cells but also sense strand DNA that binds to non-target cells that could not be completely trapped. Expected to be included. For this reason, DNA flowing out from the column 41 is amplified by real-time PCR in the same manner as in the above steps (4) to (6), and applied again to the column 41. Here, in order to increase the S / N ratio, it is preferable to perform real-time PCR in which the amount of amplification can be confirmed, and finish the reaction before reaching the plateau. This amplification by real-time PCR and the process applied to the column 41 again can be repeated as necessary. This is schematically shown on the right side of the central portion of FIG. In FIG. 6, the sense strand DNA that specifically binds to the target cell is displayed so that there is only one sense strand DNA excluding the sense strand DNA that binds to the non-target cell. It is common to be. The conditions for real-time PCR follow an already established method.
(V) Sequencing of selected DNA

  (16) Next, a sense strand DNA that specifically binds to a plurality of types of target cells selected in step (15) is inserted into a plasmid vector by TA cloning, introduced into E. coli, cloned, and amplified. To do.

  (17) Next, the sequence of the sense strand DNA that specifically binds to each target cell is determined by a normal sequence reaction.

  (18) Next, as necessary, the sequence of each sense strand DNA is bound to the target cell and the binding constant is evaluated.

  In only the first stage described above, the target cell-specific DNA finally obtained is not a single type of DNA, but may include a plurality of types of DNA as shown in the upper part of FIG. It is done. It is also conceivable that DNA having an affinity for a certain cell in the target cell but not having an affinity for another cell in the target cell can be obtained.

  Therefore, target cell-specific DNA that specifically binds to a stricter target cell is selected by the following second stage operation.

(19) The antisense strand DNA pool of the target cell-specific candidate DNA pool obtained using _one target cell (referred to as cell A1) is prepared. PCR is performed on the target cell-specific candidate DNA pool finally obtained using the cell A ₁ in the same manner as in the step (8). Primer 23 modified with biotin 24 at the 5 ′ end is used for amplification of target candidate DNA pool sense strand DNA in target cell A _1, and 5 ′ is used for amplification of target cell A ₁ specific candidate DNA pool antisense strand DNA. A primer having an amino group 25 modified at the end is used. The PCR performed here is performed according to a conventional method that has already been established.

(20) Next, after PCR, unreacted primers are removed by electrophoresis, double strands are dissociated by alkali treatment, and unnecessary target cell A _1- specific candidate DNA pool sense strand DNA 'Use a filter to remove the biotin 24 at the end. Buffer 12 ₂ containing DNA after PCR is injected into a test tube 35 having a filter 26 which does not pass through the biotin 24. Therefore, the buffer 37 that has passed through the filter 26 has only the amino group-modified antisense strand DNA of the target cell A _1- specific candidate DNA pool.

(21) Next, the antisense strand DNA of the obtained target cell A _1- specific candidate DNA pool is neutralized with dilute hydrochloric acid HCl or the like and then washed with a general reagent such as TE buffer. Thus, to return the modified target cell A ₁ specific candidate antisense strand DNA by alkali treatment for the target cell A ₁ specific candidate sense strand DNA removal.

(22) adjusts the target cell 1 cell sense strand DNA pool of target cells obtained using (a cell A ₂₎ specific candidate DNA pool. The following operation is performed using one cell (cell A ₂ ) different from the cell A ₁ described above. PCR is performed on the target cell A ₂ specific candidate DNA pool finally obtained using the cell A ₂ in the same manner as in the step (8). However here, the primers unmodified for the amplification of target cell A _2-specific candidate DNA pools sense strand DNA, biotin at the 5 'end for amplification of the target cell A _2-specific candidate DNA pool antisense strand DNA modifications Of the prepared primer. The PCR performed here is performed according to a conventional method that has already been established.

(23) After PCR, unreacted primers are removed by electrophoresis, the double strands are dissociated by alkali treatment, and biotin 24 is modified at the 5 ′ end of the unnecessary antisense strand DNA. Use a filter to remove. Buffer containing DNA after PCR is injected into the test tube 11 ₃ having a filter 26 which does not transmit biotin 24. Buffer 27 that passes through the filter 26, thus to have only the sense strand DNA of a target cell A _2-specific candidate DNA pool.

(24) Next, the sense strand DNA of the resulting target cells A _2-specific candidate DNA pool after neutralization, etc. with dilute hydrochloric acid HCl, washed with common reagents such as TE buffer. Thus, to return the modified target cell A _2-specific candidate sense strand DNA by alkali treatment for the target cell A ₁ specific candidate sense strand DNA removal.

(25) Next, the target cell A _1- specific candidate antisense strand DNA obtained in 3 above is immobilized on a carrier NHS or the like provided in a chromatography column. The immobilization of DNA on the carrier is carried out according to an established conventional method.

  (26) Next, the entire column is brought to 95 ° C., and the DNA fixed to the carrier is heat denatured.

(27) Next, the DNA of the target cell A ₂ specific candidate sense strand DNA pool obtained in 6 above is heat denatured at 95 ° C. and added to the column 41.

(28) Next, by maintaining the column 41 at the annealing temperature, the target cell A ₁ -specific candidate antisense strand DNA and the target cell A ₂ -specific candidate DNA sense strand DNA are paired to complement each other. DNA is trapped in the column 41. As a result, of the target cell A ₂ specific candidate sense strand DNA, the DNA complementary to the target cell A ₁ specific candidate antisense strand DNA is trapped in the column 41. That is, DNA that is commonly contained in the target cell A ₁ and the target cell A ₂ is trapped in the column. Conversely, DNA that is not commonly contained in the target cell A ₁ and the target cell A ₂ will flow out of the column.

  (29) Thereafter, the double strand is dissociated by alkali treatment, and the DNA paired on the column carrier is flowed out and obtained.

  (30) Next, after neutralizing the DNA with dilute hydrochloric acid HCl or the like, the DNA is restored in the same manner by washing with a general reagent such as TE buffer.

(31) The operations after step 21 are performed again on the target cell A _1- specific candidate sense strand DNA obtained in step 30. On the other hand, using the new target cell 1 cell (referred to as cell A ₃ ), the operations after step 22 are performed. By performing this similarly for other cells, a target cell-specific DNA that specifically binds to a stricter target cell is selected.

  Among the above procedures, the selection of target cells in the step (1) may be performed by using the appearance characteristics of cells based on phase contrast microscopic images or the like, for example, fluorescence instead of the method using magnetic beads with immobilized antibodies. It is good also as what isolate | separates with a cell sorter using the label | marker by. In particular, when bone marrow-derived blood cells are stained with the DNA-binding dye Hoechst 33342 in recent studies of hematopoietic stem cells, there are cells whose fluorescence intensity is smaller than that of other cells, and they are also hematopoietic. It was revealed that stem cells are present at very high concentrations. This is because hematopoietic stem cells have higher Hoechst 33342 excretion ability due to transporter activity on the cell membrane than that of other cells, and as a result, the degree of staining with Hoechst 33342 is smaller than that of other cells. ing. In this way, labeling with a fluorescent dye or the like may be used and separation may be performed with a cell sorter.

  In the process (3), only one target cell is taken out and single cell real-time PCR is performed. However, the absolute amount of DNA in the target cell specific DNA pool bound to one target cell is too small. The possibility of not being amplified by 1-cell real-time PCR is also conceivable. In that case, the number of types of DNA present in the first DNA pool may be reduced and the absolute amount of each DNA may be increased. If it is still difficult, real-time PCR may be performed by taking out a small number of cells instead of taking out only one cell.

  Further, the amplification of DNA by the normal PCR described in FIG. 5 may be real-time PCR. In this case, it is not necessary to be precise enough to amplify by the one-cell real-time PCR described in FIG.

  In addition, the method using electrophoresis for removing the primers in the steps (5) and (9) may be removed by using a filter depending on the difference in molecular weight.

  Further, the removal of the antisense strand in step (5) and the removal of the sense strand in step (9) may be performed using affinity chromatography using a column in which streptavidin is bound to a carrier.

(Example 2)
In Example 2, a nucleic acid that specifically binds to a target cell is obtained by the following procedure. The difference from Example 1 is that instead of removing DNA modified with biotin using a filter, magnetic beads to which streptavidin that binds to biotin is added are used. In the following description, the processing process (41) starts, but the processing process 50 corresponds to the processing processes (1) to (10) of the first embodiment, and 40 is added to the number assigned to the processing process of the first embodiment. It is a number.
(I) Separation of target and non-target cells

(41) an antibody that binds to the surface of the target cells prepared magnetic beads immobilized, mixed with a buffer 12 ₁ cell group are suspended, the target cell 13 and the magnetic beads 15 in which the magnetic beads 15 are bonded Separate from non-target cells 14 that did not bind. Since this process is the same as that of the first embodiment shown in FIG.

(42) FIG. 4 also shows a procedure for preparing a single-stranded DNA pool and binding to the target cell 13 in order to select a nucleic acid candidate that can be a nucleic acid that specifically binds to the target cell of the present invention. Since it is the same as that of Example 1, description and illustration are omitted.
(II) Amplification of DNA binding to target cells

(43) Next, as schematically shown in the lower left part of FIG. 7, _{one in} the buffer 12 ₂ of the test tube 11 ₁ containing the target cell 13 to which the magnetic beads 15 are bound, as schematically shown in FIG. after addition of DNA strands DNA pool, take out any one cell out of the test tube 11 _1.

(44) Next, as in Example 1, the DNA bound on the cell surface is amplified by 1-cell real-time PCR. Here, it is assumed that DNA 21 ₁ of single-stranded DNA pool is amplified. At this time, for amplification of the sense strand DNA, the primer 23 complementary to the fixed part at the 5 ′ end is used without modification. On the other hand, for amplification of antisense strand DNA, a primer 22 complementary to the fixed part at the 3 ′ end is modified with biotin 24 and used. This is schematically shown in the center of FIG.

(45) Next, after the real-time PCR, the double strand is dissociated by alkali treatment, and for the unnecessary antisense strand DNA 52, the biotin 24 is modified at its 3 ′ end to make streptavidin magnetic The bead 50 is added and removed, and the required sense strand DNA 51 is recovered. The binding between the magnetic beads 50 and the biotin 24-modified antisense strand DNA is performed according to a conventional method. This is schematically shown on the right side of FIG. Into the buffer 46 in the test tube 28 ₁ containing the DNA after the real-time PCR, the magnetic beads 50 fixed with streptavidin that binds to the biotin 24 are injected. As a result, since the biotin 24 of the biotin 24 modified antisense strand DNA 52 and the magnetic bead 50 are bonded, the DNA of the biotin 24 modified antisense strand DNA 52 is attracted to the magnet 16 when the magnet 16 is approached from the outside of the test tube 28 _1. It is done. On the other hand, the sense strand DNA 51 of the DNA remaining in the buffer 46 in the test tube 28 ₁ is transferred to the test tube 28 ₂ .

As a result, the test tube 28 ₂ has only the single-stranded DNA sense strand DNA 51 which may be DNA having a sequence that specifically binds to the target cell 13. In FIG. 7, this is indicated as a target cell-specific candidate DNA pool. In the example of the figure, four DNAs bound to the target cell 13 of the test tube 11 ₁ of Example 1 correspond.

(46) Next, the obtained target cell-specific candidate DNA pool is neutralized with dilute hydrochloric acid HCl or the like and then washed with a general reagent such as TE buffer. Thereby, the sense strand DNA denatured by alkali is recovered by alkali treatment for removing the antisense strand DNA. In FIG. 7, this process is not shown.
(III) Amplification of antisense strand DNA of DNA that binds to non-target cells

(47) Next, as shown in FIG. 3 (C), the test tube 11 ₂ in which the magnetic beads 15 antibody is immobilized is in the non-target cells 14 that did not bind, the operation (26) The DNA of the target cell-specific candidate DNA pool obtained in (1) is bound to the non-target cell 14. This is schematically shown on the left side of FIG. FIG. 8 schematically shows a state in which three DNAs among the four DNA patterns schematically shown in the target cell-specific candidate DNA pool are bound to the non-target cells 14. At this time, the DNA bound to the non-target cell 14 is a DNA having a sequence that specifically binds to the target cell 13 and may also specifically bind to the non-target cell 14.

(48) Next, collect cells 14 all in vitro 11 ₂ by centrifugal, amplifying the bound DNA on their cell surface by conventional PCR. Here, DNA21 _{2 of the} single-stranded DNA pool was amplified. At this time, a primer 22 having biotin 24 modified at the 3 ′ end is used for amplification of the antisense strand DNA, and a primer unmodified at the 5 ′ end is used for amplification of the sense strand DNA. This is schematically shown in the center of FIG. The PCR performed here is performed according to a conventional method that has already been established.

(49) Next, after PCR, the double strand is dissociated by alkali treatment, and the necessary antisense strand DNA 62 is bound to biotin 24 using the fact that biotin 24 is modified at its 3 ′ end. The streptavidin to be bound is bound to the fixed magnetic beads 50 and left in the test tube, and the sense strand DNA 61 in the buffer 46 containing the DNA after PCR is discarded. This is schematically shown on the right side of FIG. A magnetic bead 50 to which streptavidin that binds to biotin 24 is fixed is injected into the test tube 28 ₁ after PCR. When the streptavidin and biotin 24 are bonded, the magnet 16 is brought close to the test tube 28 ₁ , and the magnetic beads 50 are drawn, the antisense strand DNA 62 is drawn to the wall of the test tube 28 ₁ . In this state, the buffer and sense strand DNA 61 are discarded. Therefore, the test tube 28 ₁ has only the antisense strand DNA 62 of DNA having a sequence that specifically binds to the non-target cell 14. In FIG. 8, this was indicated as a non-target cell specific DNA pool. In the example of the figure, three of the four DNAs bound to the target cell 13 of the test tube 11 ₁ correspond.

(50) Next, the antisense strand DNA of the obtained non-target cell specific DNA pool is neutralized with HCl or the like and then washed with a general reagent such as TE buffer. Thereby, the antisense strand DNA denatured by alkali is recovered by alkali treatment for removing the sense strand DNA. In FIG. 8, this process is not shown.
(IV) Annealing on streptavidin magnetic beads

(51) Next, the sense strand DNA 51 of the target cell-specific candidate DNA pool obtained in the steps (46) and (50) and the antisense strand DNA 62 of the non-target cell-specific DNA pool with the magnetic beads 50 are obtained. Mix together in test tube 28 ₂ , heat denature at 95 ° C. and then hold at annealing temperature. As a result, among the sense strand DNAs 51 of the target cell-specific candidate DNA pool obtained in the steps (46) and (50), those complementary to the antisense strand DNA 62 of the non-target cell-specific DNA pool The sense strand DNAs 51 having these sequences can be trapped on the magnetic beads 50. When the magnet 16 is brought closer to the outside of the test tube 28 ₂ , the magnetic beads 50 are attracted to the outer wall of the test tube 28 ₂ . Therefore, the in vitro 28 ₂ buffer 46, of the target cell-specific candidate DNA pool, only DNA that its complementary strand is not included in the antisense strand DNA62 non-target cell-specific DNA is left. This is expressed as a sense strand pool of target cell-specific DNA, and is schematically shown at the bottom of FIG.

(52) However, the DNA remaining in the test tube 28 _{and second} buffer 46, still not only DNA that specifically binds to the target cells, DNA completely bound to non-target cells that could not be traps Expected to be included. Therefore, it said left in the test tube 28 _{and second} buffer 46 DNA Process (24) - (26) and amplified by the same real-time PCR, again, antisense non-target cell-specific DNA dated magnetic beads 50 The DNA strand 62 and the test tube 28 ₂ are put together and trapped on the magnetic bead 50. Here, in order to increase the S / N ratio, it is preferable to perform real-time PCR in which the amount of amplification can be confirmed, and finish the reaction before reaching the plateau. The amplification by the real-time PCR and the process of trapping again on the magnetic beads 50 can be repeated as necessary. This is schematically shown on the right side of the central portion of FIG. In FIG. 9, the DNA that specifically binds to the target cell is shown as having only one DNA excluding the DNA that binds to the non-target cell, but this is generally multiple types. is there. The conditions for real-time PCR follow an already established method.
(V) Sequencing of selected DNA

  (53) Next, a plurality of types of DNA selected in the step (52) are inserted into a plasmid vector by the TA cloning method, introduced into E. coli, cloned and amplified.

  (54) Next, the sequence of each DNA is determined by a normal sequence reaction.

  (55) Next, if necessary, each DNA sequence is bound to a target cell and the binding constant is evaluated.

  Also in Example 2, target cell-specific DNA that specifically binds to a stricter target cell is selected by the following second stage operation.

(56) The antisense strand DNA pool of the target cell-specific candidate DNA pool obtained using _one target cell (referred to as cell A1) is prepared. PCR is performed on the target cell-specific candidate DNA pool finally obtained using the cell A ₁ in the same manner as in the step (48). At this time, for amplification of the antisense strand DNA, a primer 22 modified with biotin 24 at the 5 ′ end is used, and for amplification of the sense strand DNA, a primer unmodified at the 5 ′ end is used. The PCR performed here is performed according to a conventional method that has already been established.

(57) Same as steps (49) and (50). Thereby, the antisense strand DNA of the target cell A _1- specific candidate DNA pool is immobilized on the surface of the streptavidin magnetic beads.

(58) The sense strand DNA pool of the target cell-specific candidate DNA pool obtained using another target cell 1 cell (referred to as cell A ₂ ) is prepared. The following operation is performed using one cell (cell A ₂ ) different from the cell A ₁ described above. PCR is performed on the target cell A ₂ specific candidate DNA pool finally obtained using the cell A ₂ in the same manner as in the step (48). However here, the primers unmodified for the amplification of target cell A _2-specific candidate DNA pools sense strand DNA, biotin at the 5 'end for amplification of the target cell A _2-specific candidate DNA pool antisense strand DNA modifications Of the prepared primer. The PCR performed here is performed according to a conventional method that has already been established.

(59) After the PCR, in the same manner as in Process (45), to remove the anti-sense strand DNA of which unnecessary target cell A _2-specific candidate DNA pool, only the sense strand DNA of a target cell A _2-specific candidate DNA pool It will have.

(60) Then, similarly to the step (46), after neutralization sense strand DNA of the resulting target cells A _2-specific candidate DNA pool and with dilute hydrochloric acid HCl, washed with common reagents such as TE buffer . Thereby, the target cell A ₂ specific candidate sense strand DNA denatured by alkali treatment for removing the target cell A ₂ specific candidate sense strand DNA is restored.

(61) Next, annealing is performed on streptavidin magnetic beads as in the step (51). A target cell A ₁ specific candidate antisense strand DNA, that allow pairing sense strand DNA of a target cell A _2-specific candidate DNA, thereby trapping the complementary DNA. As a result, of the target cell A ₂ specific candidate sense strand DNA, DNA complementary to the target cell A ₁ specific candidate antisense strand DNA is trapped. That is, the DNA commonly contained in the target cell A ₁ and the target cell A ₂ is trapped. Conversely, DNA that is not commonly contained in the target cell A ₁ and the target cell A ₂ is not trapped and remains in the supernatant and is removed by washing.

(62) Thereafter, the target strand A _1- specific candidate sense strand DNA which has been dissociated by alkali treatment and paired with the target cell A-specific candidate antisense strand DNA immobilized on the streptavidin magnetic beads To get.

  (63) Next, after neutralizing the DNA with dilute hydrochloric acid HCl or the like, the DNA is recovered in the same manner by washing with a general reagent such as TE buffer.

  (64) The operation from step 1 is performed again on the target cell A-specific candidate sense strand DNA obtained in step 63. On the other hand, using the new target cell 1 cell (referred to as cell C), the operations after step 4 are performed. By performing this similarly for other cells, a target cell-specific DNA that specifically binds to a stricter target cell is selected.

  Also in Example 2, the selection of target cells in the step (41) in the above procedure uses the appearance characteristics of the cells, for example, labeling by fluorescence instead of the method using magnetic beads with immobilized antibodies. Then, it may be separated by a cell sorter.

  Further, instead of extracting only one target cell, real-time PCR may be performed by extracting a small number of cells.

  In addition, the amplification of DNA by the normal PCR described in FIG. 8 may be real-time PCR, but the precision required to amplify by the one-cell real-time PCR described in FIG. 7 is not required. And it is sufficient.

  Further, the method using electrophoresis for removing the primers in the steps (45) and (49) may be removed by using a filter depending on the difference in molecular weight.

  Further, the removal of the antisense strand in the step (45) and the removal of the sense strand in the step (49) may be performed by affinity chromatography using a column in which streptavidin is bound to a carrier.

  While exemplary embodiments of the present invention have been described above, various changes, modifications, and improvements will readily occur to those skilled in the art. The foregoing is merely illustrative of the principles of this invention and various modifications can be made by those skilled in the art without departing from the scope and spirit of the invention.

  According to the present invention, a DNA that specifically binds to a target cell and does not bind to a non-target cell can be easily obtained, so that it can contribute to research development in the drug discovery and medical fields.

(A)-(F) is a flowchart explaining the 1st stage which implements this invention. (A), (B) is a flowchart explaining the 2nd stage which implements this invention. (A), (B), and (C) schematically show a procedure for performing separation of target cells and non-target cells of Example 1 using magnetic beads on which an antibody that binds to the cell surface is immobilized. FIG. FIG. 3 is a diagram schematically showing a procedure for selecting a target cell-specific candidate DNA of Example 1. FIG. 3 is a diagram schematically showing a procedure for selecting an antisense strand DNA of a DNA that binds to a non-target cell among the target cell-specific candidate DNAs of Example 1. It is a figure which shows typically the procedure which removes the DNA couple | bonded with a non-target cell from affinity cell specific candidate DNA by affinity chromatography, and refine | purifies DNA. It is a figure which shows typically the procedure which selects the target cell specific candidate DNA of Example 2. FIG. It is a figure which shows typically the procedure which selects the antisense strand DNA of DNA couple | bonded with the target cell among the target cell specific candidate DNA of Example 2. FIG. It is a figure which shows typically the procedure which removes the DNA couple | bonded with the target cell among target cell specific candidate DNA by annealing on a streptavidin magnetic bead, and refine | purifies DNA.

Explanation of symbols

11 ₁ , 11 ₂ , 11 ₃ , 28 ₁ , 28 ₂ , 35 ... test tube, 12 ₁ , 12 ₂ , 27, 37, 46 ... buffer, 13 ... target cell, 14 ... non-target cell, 15 ... antibody fixed Magnetic beads, 16 ... magnets, 21 ₁ , 21 ₂ , --n, 21 _n ... DNA, 22, 23 ... primers, 24 ... biotin, 25 ... amino groups, 26 ... filters, 41 ... chromatography columns , 42 ... carrier in the column, 50 ... streptavidin magnetic beads, 51, 61 ... sense strand DNA, 52, 62 ... antisense strand DNA.

Claims (15)

(I) separating the target cell group and the non-target cell group from the cell group;
(Ii) applying a single-stranded DNA pool to target cells to bind single-stranded DNA that specifically binds to the target cells;
(Iii) a target cell-specific candidate DNA pool comprising target cell-specific candidate DNAs that are selected from the target cells bound to the specifically-bound single-stranded DNA and bound to the selected target cells The process of creating,
(Iv) applying the target cell-specific candidate DNA pool to non-target cells to bind the target cell-specific candidate DNA that specifically binds to the non-target cells to the non-target cells;
(V) creating a non-target cell-specific DNA pool comprising target cell-specific candidate DNAs bound to the non-target cells to which target cell-specific candidate DNAs that specifically bind to the non-target cells are bound And (vi) removing from the target cell-specific candidate DNA obtained in step (iii) a DNA complementary to the non-target cell-specific DNA obtained in step (v) by annealing. Determining a target cell-specific DNA,
A method for selecting a nucleic acid that specifically binds to a target cell. Further comprising the step of performing the steps (i) to (vi) on different target cells,
A DNA having a nucleic acid sequence that matches with respect to all of the target cell-specific DNA pools by contrasting each target cell-specific DNA pool containing the target cell-specific DNA determined for each of the different target cells The nucleic acid selection method according to claim 1, wherein is determined as target cell-specific DNA for the target cell group. (I) separating the target cell group and the non-target cell group from the cell group;
(Ii) applying a single-stranded DNA pool to target cells to bind single-stranded DNA that specifically binds to the target cells;
(Iii) The target cell to which the specifically bound single-stranded DNA is bound is selected, and the single-stranded DNA bound to the selected target cell is used as the sense strand DNA and bound. Amplifying single-stranded DNA by one-cell real-time PCR;
(Iv) selecting a sense strand DNA of the amplified single-stranded DNA to create a target cell-specific candidate sense strand DNA pool;
(V) Applying the target cell-specific candidate sense strand DNA pool to the non-target cell to bind the target cell-specific candidate sense strand DNA that specifically binds to the non-target cell to the non-target cell Process,
(Vi) The target cell-specific candidate sense strand DNA bound to the non-target cell to which the target cell-specific candidate sense strand DNA that specifically binds to the non-target cell is bound as the sense strand DNA as the target Amplifying the cell-specific candidate sense strand DNA by PCR;
(Vii) selecting the antisense strand DNA of the amplified target cell-specific candidate sense strand DNA to create a non-target cell-specific antisense strand DNA pool; and (viii) obtained in step (iv) Removing the DNA complementary to the non-target cell-specific antisense strand DNA obtained in step (vii) from the target cell-specific candidate sense strand DNA by annealing,
A method for selecting a nucleic acid that specifically binds to a target cell. The step (i)
Applying magnetic beads on which antibodies that bind to the target cells are immobilized to the cell group to magnetically separate the target cells to which the magnetic beads are bound;
The nucleic acid selection method of Claim 3 containing this. The step (vi)
Amplifying the target cell-specific candidate sense strand DNA by real-time PCR;
The nucleic acid selection method of Claim 3 containing this. In the steps (iii) and (iv),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is removed by a filter, and only a single-stranded DNA sense strand DNA is selected to create a target cell-specific candidate sense strand DNA pool.
The nucleic acid selection method according to claim 3. In the steps (iii) and (iv),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is magnetically removed using streptavidin magnetic beads that bind to biotin, and only the single strand DNA sense strand DNA is selected, and the target cell specific candidate sense strand DNA is selected. A pool is created,
The nucleic acid selection method according to claim 3. In the steps (iii) and (vi),
Primer for amplification of sense strand DNA is modified with biotin, primer for amplification of antisense strand DNA is modified with amino group,
The amplified sense strand DNA is removed by a filter to create an antisense strand DNA pool of non-target cell specific candidate sense strand DNA pools.
The nucleic acid selection method according to claim 3. In the steps (iii) and (vi),
Primer for amplification of antisense strand DNA is modified with biotin, primer for amplification of sense strand DNA is unmodified,
The amplified antisense strand DNA is left magnetically using a streptavidin magnetic bead that binds to biotin, and non-target cell-specific candidate sense strand DNA is removed to obtain a non-target cell-specific candidate sense strand. An antisense strand DNA pool of the DNA pool is created;
The nucleic acid selection method according to claim 3. Said step (viii)
Fixing the antisense strand DNA of the non-target cell-specific DNA to a carrier provided in a chromatography column; and introducing the target cell-specific candidate sense strand DNA into the chromatography column; Removing the target cell-specific candidate sense strand DNA complementary to the antisense strand DNA of the non-target cell-specific DNA by annealing;
The nucleic acid selection method of Claim 8 containing this.   The nucleic acid selection method according to claim 10, comprising amplifying the target cell-specific candidate sense strand DNA flowing out from the chromatography column by real-time PCR and repeatedly introducing it into the chromatography column. Said step (viii)
The target cell-specific candidate sense strand DNA is introduced into the magnetically left antisense strand DNA using the streptavidin magnetic beads that bind to the biotin, and the non-target cell-specific DNA antisense strand DNA Removing the complementary target cell-specific candidate sense strand DNA by annealing;
The nucleic acid selection method according to claim 9, comprising:   The target cell-specific candidate sense strand DNA that has been removed by annealing the target cell-specific candidate sense strand DNA complementary to the antisense strand DNA of the non-target cell-specific sense strand DNA is amplified by real-time PCR. The target cell-specific candidate sense strand DNA is repeatedly introduced into the magnetically left antisense strand DNA using the streptavidin magnetic beads that bind to the biotin, and the non-target cell-specific DNA antisense strand The nucleic acid selection method according to claim 12, comprising removing the target cell-specific candidate sense strand DNA complementary to the DNA by annealing. Further comprising performing the steps (i) to (viii) on different target cells,
A DNA having a nucleic acid sequence that matches with respect to all of the target cell-specific DNA pools by contrasting each target cell-specific DNA pool containing the target cell-specific DNA determined for each of the different target cells The nucleic acid selection method according to claim 3, wherein is determined as a target cell-specific DNA for the target cell group.   A method for producing a nucleic acid that specifically binds to a target cell, comprising the step of using the nucleic acid selection method according to claim 1.

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