JP4395331B2 - Nucleic acid detection method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a nucleic acid detection method for detecting a DNA complex formed by binding double-stranded DNA and single-stranded DNA.
[0002]
[Prior art]
  Conventionally, a method for forming a complex containing double-stranded DNA and single-stranded DNA is known. Specifically, a target DNA (double-stranded DNA) is prepared, and a probe DNA (single-stranded DNA) having a base sequence complementary to a partial base sequence of one of the DNA strands of the target DNA is prepared. . Next, these DNA and Escherichia coli (Escherichia coli) RecA protein is mixed and kept warm for a sufficient time. As a result, a DNA-protein complex composed of the target DNA, the probe DNA, and the RecA protein is formed. Specifically, first, the RecA protein binds to the probe DNA to form a probe DNA-RecA protein complex. Subsequently, the probe DNA-RecA protein complex binds to the target DNA, and a DNA-protein complex having a triplex forming region is formed. At this time, the probe DNA is considered to bind to a region consisting of a base sequence complementary to the probe DNA in the target DNA with the RecA protein involved. The DNA-protein complex in this state is relatively stable.
[0003]
  However, when this DNA-protein complex is added with sodium dodecyl sulfate (SDS) or a proteolytic enzyme (for example, proteinase K) and incubated for a sufficient period of time to inactivate the RecA protein, the DNA-protein complex Not only is the RecA protein removed, but the binding between the target DNA and the probe DNA is also eliminated. That is, the structure of the DNA-protein complex was stably maintained only in the presence of the RecA protein, and a stable DNA complex could not be formed without the RecA protein. In a field such as genetic engineering, when a DNA complex is to be used, a complex in a state where the RecA protein is bound receives many limitations due to the presence of the RecA protein, and the range of use becomes narrow. Thus, development of a method for forming a DNA complex capable of maintaining a stable structure even in the absence of such a protein has been awaited.
[0004]
  In order to solve such a problem, the present applicant has found a method for forming a DNA complex that can maintain a stable state despite the fact that the complex does not contain a protein (see Patent Document 1). That is, a linear single-stranded DNA comprising a linear double-stranded DNA and a base sequence substantially complementary to the base sequence starting from the base near the 5 ′ end of one of the double-stranded DNAs. Prepare double-stranded DNA. Next, these DNA and Escherichia coli (Escherichia coli) RecA protein, and then E. coli (Escherichia coli) Exonuclease I) and incubate for sufficient time. As a result, a relatively stable DNA-protein complex including at least double-stranded DNA, single-stranded DNA, and RecA protein is formed. The DNA-protein complex thus formed can form a stable DNA complex even if the recombinant protein and nuclease are subsequently inactivated. That is, it is possible to form a DNA complex that can maintain a stable state despite the fact that no protein is contained in the complex.
[Problems to be solved by the invention]
  For example, if a single-stranded DNA is labeled in advance, such a DNA complex is labeled. Therefore, the presence of the DNA complex can be confirmed by a technique applying Southern hybridization.
  However, since the DNA complex could not be visualized and confirmed conventionally, the structure and form of the DNA complex could not be examined. In addition, since a technique using Southern hybridization detects a population (bulk) of DNA complexes, the DNA complex cannot be reliably detected when the amount of the sample is small. Moreover, there existed problems, such as requiring time for a detection.
[0005]
  The present invention has been made in view of the present situation, and an object thereof is to provide a nucleic acid detection method capable of visualizing and detecting a DNA complex in which double-stranded DNA and single-stranded DNA are bound.
[0006]
[Means for Solving the Problems]
  The solution is a linear double-stranded DNA, a base sequence starting from the base near the 5 'end of one of the double-stranded DNAs.,phaseLinear single-stranded DNA containing a complementary base sequence,It is RecA protein of Escherichia coliRecombinant protein and E. coli (Escherichia coli) ExonucleaseIA certain nuclease is reacted, and the above-mentioned double-stranded DNA has the complementary base sequence of the single-stranded DNA in the near-terminal inclusion region including the vicinity of the 5 ′ end of the one DNA strand. A DNA-protein complex forming step in which a complementary region forms a DNA-protein complex formed by binding in a state where at least the recombinant protein is involved; and the recombinant protein and the nuclease are inactivated, A protein deactivation step for forming a DNA complex formed by binding the complementary region of the single-stranded DNA to a region near the end of the single-stranded DNA, and AFM detection for detecting the DNA complex by an atomic force microscope And a processIn the DNA-protein complex forming step, ATP-γS is added, and after the protein deactivation step and before the AFM detection step, a nuclease treatment step for reacting the DNA complex with the nuclease is provided.This is a method for detecting a nucleic acid.
[0007]
  According to the present invention, in the DNA-protein complex formation step, a DNA-protein complex is formed from double-stranded DNA, single-stranded DNA, recombinant protein, and nuclease. Then, in the protein inactivation step, when the recombinant protein and nuclease are inactivated, a DNA complex in which the complementary region of the single-stranded DNA is bound to the region near the end of the double-stranded DNA can be formed. Such a DNA complex is stable without being dissociated even if a certain amount of heat is applied even if there is no special protein or the like (for example, the RecA protein described above) for stabilizing the structure. Can be maintained. Thereafter, in the AFM detection step, this DNA complex is detected by an atomic force microscope (AFM).
[0008]
  If the DNA complex is detected by such a method, the DNA complex can be visualized and confirmed, so that the structure and form of the DNA complex can also be examined. Moreover, since each DNA complex is visualized, the DNA complex can be reliably detected even if the amount of the sample is small. Furthermore, as compared with the technique applying Southern hybridization, it is not necessary to label a part of DNA, and it is not necessary to perform electrophoresis of the DNA complex or to dry the gel. It takes less time.
  In addition, the binding region of double-stranded DNA and single-stranded DNA may be formed not only in the inclusion region near one end of double-stranded DNA, but also in the inclusion region near both ends of double-stranded DNA. Is possible.
  In view of availability, safety, and functionality, it is preferable to use a RecA protein derived from E. coli.
  In addition, in the DNA-protein complex formation step, if ATP-γS is added and reacted, a DNA protein complex can be formed more efficiently. As a result, a DNA complex can be formed more efficiently.
  In addition, when ATP-γS is added in the DNA-protein complex formation step to form a DNA protein complex, a DNA complex is formed in which part of one of the double-stranded DNA strands is released in a whisker-like manner. Sometimes. In contrast, in the present invention, since the DNA complex and nuclease are reacted after the protein deactivation step and before the AFM detection step, the DNA released in a whisker-like manner is degraded by the nuclease. Accordingly, a DNA complex having no free beard-like portion can be observed by AFM.
[0009]
  Here, the binding form of the DNA conjugate is not limited as long as the complementary region of the single-stranded DNA is bound to the inclusion region near at least one end of the double-stranded DNA. That is, the double-stranded DNA and the single-stranded DNA do not necessarily have to have a specific structure such as a Watson-Crick base pair or a Hoogsteen-type base pair. What is necessary is just to be couple | bonded by some specific interaction between the region near the end and the complementary region of the single-stranded DNA.
[0010]
  Any double-stranded DNA may be used as long as it is linear. That is, it may consist of any base sequence, and theoretically there is no upper limit to their chain length. Therefore, for example, it may be a huge DNA having the full length of the human genome called 3000 Mbp. Of course, the origin of double-stranded DNA is not questioned. Therefore, even if it is DNA derived from the genome of viruses, microorganisms, animals and plants or modified DNA thereof, even plasmid DNA etc. possessed by microorganisms, chimeric DNA obtained by inserting a heterologous DNA fragment into plasmid DNA, etc. Alternatively, it may be an artificially synthesized oligonucleotide or the like.
[0011]
  Single-stranded DNA is any linear DNA that includes a base sequence starting from the base near the 5 'end of one of the double-stranded DNA and a substantially complementary base sequence. It may be used. That is, any base sequence may be used as long as this condition is satisfied, and there is theoretically no upper limit to their chain length. Moreover, the origin is also not asked. These points are the same as the double-stranded DNA.
[0012]
ComplementaryThe degree is generally required to have approximately 70-80% or more complementarity, and more preferably approximately 100% complementarity. This is because the higher the complementarity, the more stable DNA complex can be formed. However, the required degree of complementarity varies slightly depending on the length of the complementary region. In addition, even if the complementarity of the entire complementary region is the same (for example, 70%), the complementary region has almost the same complementarity (70%). (For example, 90%) and a low region (for example, 50%) are unevenly distributed.
[0013]
  In addition, since single-stranded DNA only needs to contain a complementary region, even if the entire single-stranded DNA consists of a complementary region, or another base sequence is added to the complementary region. It may be the one that was made. However, it is preferable that the entire single-stranded DNA consists of complementary regions because a more stable DNA complex can be easily formed.
  The single-stranded DNA must contain a base sequence that is substantially complementary to the “base sequence starting from the base near the end” of one of the double-stranded DNAs as follows. For a reason. That is, when the single-stranded DNA is complementary only to the base sequence other than the vicinity of the end of one DNA strand of the double-stranded DNA (for example, the base sequence near the center), a DNA-protein complex formation step Can form a stable DNA-protein complex, but if the protein is deactivated in the protein deactivation process, the double-stranded DNA and the single-stranded DNA are dissociated, and a stable DNA complex cannot be obtained. It is.
[0014]
  Similarly, the single-stranded DNA must contain a base sequence that is substantially complementary to the base sequence starting from the base “near the 5 ′ end” of one of the double-stranded DNAs. For a reason. That is, when the single-stranded DNA is complementary only to the base sequence “near the 3 ′ end” of one of the double-stranded DNAs, a stable DNA- This is because a protein complex can be formed, but when the protein is inactivated, the double-stranded DNA and the single-stranded DNA are dissociated, and a stable DNA complex cannot be obtained.
[0015]
[0016]
[0017]
[0018]
  The DNA-protein complex formation step is preferably performed in a buffer in the presence of nucleoside triphosphate or an analog thereof. This is because a stable DNA-protein complex can be formed efficiently.
  The buffer solution can be appropriately changed depending on the recombinant protein and nuclease to be used in order to obtain optimum reaction conditions. For example, use a Tris buffer with a pH adjusted to about 4.0 to 9.0, more preferably about 7.0 to 8.0 with tris (tris (hydroxymethyl) aminomethane) and a suitable acid such as acetic acid or hydrochloric acid. That's fine. The buffer is generally used at about 10-100 mM, preferably about 30 mM.
[0019]
  Examples of nucleoside triphosphates or analogs thereof include adenosine 5′-triphosphate (ATP), guanosine 5′-triphosphate (GTP), UTP, CTP, and adenosine (γ-thio) -triphosphate. (ATP-γS), guanosine (γ-thio) -triphosphate (GTP-γS), dATP, dUTP, dCTP, and the like. They can also be used in combination with nucleoside diphosphates (eg ADP). These nucleoside triphosphates and the like are used at 0.1 to 10 mM, preferably about 5 mM.
[0020]
  The concentration of the nucleic acid (double-stranded DNA and single-stranded DNA) in the reaction solution may be any concentration that can sufficiently dissolve the nucleic acid, and can be changed as appropriate. The ratio of single-stranded DNA to double-stranded DNA is preferably about 1 to 100 times in molar ratio. In addition, the recombinant protein is preferably added so as to correspond to one molecule per three bases of single-stranded DNA. However, the optimal amount varies slightly depending on the recombinant protein used. The nuclease is preferably added in an amount of about 1 unit per 1 μg of double-stranded DNA. However, the optimum amount also varies slightly depending on the nuclease used.
  The reaction solution prepared as described above can form a DNA-protein complex by incubating at 4 to 60 ° C., preferably about 37 ° C., for 5 minutes or longer, generally about 30 to 60 minutes.
[0021]
  In this step, in addition to the method of adding all the components to the reaction solution as described above and keeping the temperature constant for a certain period of time, the following method may be employed. That is, first, double-stranded DNA, single-stranded DNA and recombinant protein are added to a buffer containing nucleoside triphosphate and the like, and kept at 4 to 60 ° C., preferably about 37 ° C. for several minutes or more. Incubate for about 10 minutes. Thereafter, a nuclease is added to the reaction solution, and the mixture is further kept at 4 to 60 ° C., preferably about 37 ° C., for 5 minutes or longer, generally about 30 minutes.
[0022]
  When reacted by such a method, a particularly stable DNA-protein complex can be formed. The reason for this is that exonuclease I and the like are originally enzymes that cleave single-stranded DNA from the end, so that when added to the reaction solution from the beginning, the single-stranded DNA may be scraped. However, when exonuclease I or the like is added later in this way, the single-stranded DNA is less likely to be cleaved because the recombinant protein is first bound to the single-stranded DNA and protected from exonuclease I. This is because it is considered that a stable DNA-protein complex can be formed.
[0023]
  In order to inactivate the protein in the protein deactivation step, a chelating agent (for example, ethylenediaminetetraacetic acid), sodium dodecyl sulfate (SDS), or a proteolytic enzyme (for example, proteinase K) is added to the reaction solution. ) Or after a combination of these treatments, for example, it may be kept at about 37 ° C. for 10 minutes or more. In order to recover only the DNA complex from the reaction solution thus obtained, a method of recovering by appropriate column chromatography after deproteinization treatment by solvent extraction with phenol, chloroform or the like, or ethanol precipitation The method of precipitating DNA may be used once.
[0024]
[0025]
[0026]
[0027]
[0028]
  Furthermore, the acid detection method according to any one of the above, wherein the nucleic acid detection method is characterized in that nucleoside triphosphate is added in the DNA-protein complex formation step.
[0029]
  Thus, in the DNA-protein complex formation step, if a nucleoside triphosphate such as ATP is added and reacted, a DNA protein complex can be formed more efficiently. As a result, a DNA complex can be formed more efficiently.
[0030]
[0031]
[0032]
[0033]
[0034]
  Furthermore, the nucleic acid detection method according to any one of the above, wherein the single-stranded DNA has a nucleotide sequence of 60 mer or more.
[0035]
  Single-stranded DNA can bind to double-stranded DNA to form a DNA complex even if its chain length is short. However, if the single-stranded DNA is short, specifically, shorter than 60 mer, it is difficult to distinguish the double-stranded DNA from the DNA complex when observed with an atomic force microscope. That is, it becomes more difficult to detect the DNA complex.
  In contrast, in the present invention, single-stranded DNA having a base sequence of 60 mer or more is used. Therefore, the DNA complex can be detected more reliably when observed with an atomic force microscope.
[0036]
DETAILED DESCRIPTION OF THE INVENTION
(Embodiment 1)
  Embodiments of the present invention will be described below with reference to the drawings.
  First, target DNA (double-stranded DNA) is prepared (see FIG. 1). FIG. 1 schematically shows the formation of a DNA complex. In this embodiment, pSP64poly (A) DNA (Promega) (3030 bp) is cleaved into three DNA fragments (1892 bp, 676 bp and 462 bp DNA fragments) with the restriction enzyme RsaI, and the largest DNA fragment (about 1.9 kbp). ) As the target DNA. Specifically, pSP64poly (A) DNA was cleaved with the restriction enzyme RsaI, and the target DNA fragment was separated and purified using gel filtration. For gel filtration, S-1000 Sephacryl (Amersham Marmacia Co., Ltd.) was used, and TE Buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1.5 M NaCl) was used as an elution buffer on a 100 cm open column. went. 2 ml of the eluted DNA sample solution was collected at a rate of 1 mL / min. Then, a part of each collected sample was confirmed by electrophoresis. The result is shown in FIG.
[0037]
  As is clear from FIG. 2, almost no signal was detected around lanes 1 and 2. In addition, in lanes 3 to 20, a signal derived from the largest DNA fragment of about 1.9 kbp was mainly confirmed. In addition, in lanes 21 to 27, the signal derived from the second largest DNA fragment of about 0.7 kbp was mainly confirmed, together with the signal derived from the largest fragment of about 1.9 kbp. In addition, in lanes 28 to 29, signals derived from the second largest DNA fragment of about 0.7 kbp were mainly confirmed. In Lanes 30 to 37, a signal derived from the second largest DNA fragment of about 0.7 kbp and a signal derived from the smallest DNA fragment of about 0.5 kbp were mainly confirmed. Based on these results, the samples used in lanes 3 to 20 were used as the target DNA.
[0038]
  Next, probe DNA (single-stranded DNA) is prepared (see FIG. 1). In this embodiment, five types of probe DNAs having different lengths, that is, a first probe DNA of 150mer, a second probe DNA of 300mer, a third probe DNA of 600mer, a fourth probe DNA of 900mer, and a first probe DNA of 1200mer. Five probe DNAs were prepared. Specifically, the shortest first probe DNA was prepared as oligonucleotide 1 by DNA synthesis. On the other hand, the 2nd probe DNA-the 5th probe DNA were prepared using PCR reaction. The PCR reaction was carried out using 600 ng of pSP64poly (A) DNA (Promega) as a template in 50 μl of PCR reaction solution, 0.5 mM (final concentration) forward primer DNA and 0.5 mM (final concentration) reverse primer DNA. , 0.25 unit TaKaRa Taq (Takara Bio Inc.), 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP were mixed with 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2. . The PCR reaction was performed under the conditions of 1 cycle (94 ° C., 2 min), 30 cycles (94 ° C., 30 sec-60 ° C., 30 sec-74 ° C., 60 sec), 1 cycle (74 ° C., 7 min-4 ° C., 1 min). . Thereafter, a dye solution was added to the PCR reaction solution, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the agarose gel was stained with ethidium bromide and a photograph of the electrophoresis gel was recorded. The results are shown in lanes 1 to 4 in FIG.
[0039]
  For the second probe DNA, oligonucleotide 2 was used as the forward primer DNA and oligonucleotide 3 was used as the reverse primer DNA. In the third probe DNA, oligonucleotide 4 was used as the forward primer DNA, and oligonucleotide 3 was used as the reverse primer DNA. In the fourth probe DNA, oligonucleotide 5 was used for the forward primer DNA and oligonucleotide 3 was used for the reverse primer DNA. In the fifth probe DNA, oligonucleotide 6 was used as the forward primer DNA and oligonucleotide 3 was used as the reverse primer DNA. Oligonucleotide 1 to oligonucleotide 6 are all composed of a sequence that is 100% complementary to pSP64poly (A) DNA. In addition, biotin was added to the 5 ′ end of the oligonucleotide 3 in order to prepare a single-stranded probe DNA later.
[0040]
Oligonucleotide 1:
5'-tgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagt-3 '
Oligonucleotide 2:
5'-gccgggaagctagagtaagtagtt-3 ’
Oligonucleotide 3:
5'-actcaccagtcacagaaaagcatc-3 ’
Oligonucleotide 4:
5'-gttttaaatcaatctaaagtatat-3 ’
Oligonucleotide 5:
5'-ggtggcctaactacggctacacta-3 ’
Oligonucleotide 6:
5'-tcgtgcgctctcctgttccgaccc-3 '
[0041]
  As is clear from FIG. 3, in lane 1, a signal derived from a 300 bp target DNA fragment was strongly detected. In lane 2, a signal derived from a 600 bp target DNA fragment was strongly detected. In lane 3, a signal derived from the 900 bp target DNA fragment was strongly detected. In lane 4, a signal derived from a 1200 bp target DNA fragment was strongly detected.
[0042]
  Next, the target PCR product was separated and purified from the PCR reaction solution used in Lane 1 by gel filtration. Gel filtration was performed on a 20 cm open column using S-400 Sephacryl (Amersham Marmacia Co., Ltd.) and TE Buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1.5 M NaCl) as the eluent. It was. Thereafter, a dye solution was added to the purified DNA solution, and electrophoresis was performed on a 1% agarose gel. After the electrophoresis, the agarose gel was stained with ethidium bromide, and a photograph of the electrophoresis gel was recorded. Similarly, the target PCR products were separated and purified from the PCR reaction solutions used in lanes 2 to 4 by gel filtration, and electrophoresis was performed. The results are shown in lanes 5 to 8 in FIG.
[0043]
  As is clear from FIG. 3, in lane 5, a signal derived from the target DNA fragment of 300 bp was strongly detected, and other signals were decreased as compared with lane 1. Further, in lane 6, a signal derived from the 600 bp target DNA fragment was strongly detected, and other signals were decreased as compared with lane 2. In lane 7, a signal derived from the 900 bp target DNA fragment was strongly detected, and other signals were decreased from those in lane 3. Further, in lane 8, a signal derived from a 1200 bp target DNA fragment was strongly detected, and other signals were decreased from those in lane 4. Therefore, by subjecting the PCR reaction solution to gel filtration, the target PCR product could be separated and purified with higher purity.
[0044]
  Next, single-stranded probe DNA (second probe DNA to fifth probe DNA) was prepared from each PCR product that was double-stranded. Specifically, 125 ul (10 ug / ul) of Dynabeads Streptavidin (DYNAL) was washed once with 500 ul of 6XSSC buffer using a magnetic stand and suspended in 125 ul of 6XSSC buffer. The supernatant was removed, and 50 ul of PCR amplification product that had been purified by gel filtration diluted in 125 ul of 6XSSC buffer was mixed therewith. The binding reaction between the PCR product and Dynabeads Streptavidin was performed at room temperature for 60 minutes. After removing the supernatant using a magnet stand, it was washed 3 times with 500 ul of 6XSSC buffer and once with 250 ul of 1XSSC buffer to remove unreacted PCR product. For denaturation of single-stranded DNA, 100ul of NaOH (0.15M) was added and reacted at room temperature for 10 minutes, and the supernatant was transferred to a new tube using a Magnet stand. 11ul 10XTE buffer (pH7.5), 6.5ul Acetic scid (1.25M) was added thereto, and ethanol precipitation was performed to concentrate and recover single-stranded DNA.
[0045]
  The first probe DNA consists of a 150-mer base sequence that is 100% complementary to a 150-mer base sequence starting from the 5′-end base of one DNA strand of the target DNA. The second probe DNA consists of a 300-mer base sequence that is 100% complementary to a 300-mer base sequence starting from the 5′-end base of one DNA strand of the target DNA. The third probe DNA is composed of a 600mer base sequence that is 100% complementary to a 600mer base sequence starting from the 5 ′ end base of one DNA strand of the target DNA. The fourth probe DNA consists of a 900-mer base sequence that is 100% complementary to a 900-mer base sequence starting from the 5′-end base of one DNA strand of the target DNA. The fifth probe DNA is composed of a 1200mer base sequence starting from the 5 ′ end base of one DNA strand of the target DNA and a 1200mer base sequence which is 100% complementary.
[0046]
  Here, the prepared double-stranded target DNA and single-stranded probe DNA were observed with an atomic force microscope (AFM). The procedure for preparing the AFM specimen was as follows. First, magnesium chloride is added to a DNA solution containing double-stranded target DNA and single-stranded third probe DNA to a final concentration of 10 mM, and 50 μl is dropped onto a fresh surface immediately after cleavage of a 10 mm square mica substrate. And allowed to stand at room temperature for 5 minutes. Next, this was washed with 5 ml of sterilized water, water was blown off with nitrogen gas, and further dried under vacuum at 40 ° C. for 15 minutes. This specimen was observed with an atomic force microscope. DNA visualization conditions were measured in the tapping mode using a silicon single crystal cantilever (D-NCH-10V) with the sample prepared above set on a scanning probe microscope (Nanoscope IIIa) (Degital Instruments). did. The scan range was 0.5-2 μm in the XY direction and 2 nm in the height (Z-axis) direction. DNA length was measured using NIHimage, a freeware. The result is shown in FIG. Moreover, what showed the photograph of FIG. 4 typically is shown in FIG.
[0047]
  As apparent from FIG. 4, according to the atomic force microscope, it was possible to reliably observe the state in which each double-stranded DNA of about 1.9 kbp was linearly present (left side in the figure). In addition, the presence of the 600-mer single-stranded third probe DNA was reliably observed for each molecule (upper right in the figure). In single-stranded DNA, thickened and granular parts can be seen in some places. This is because single-stranded DNA has a higher order structure by binding complementary sequences within the same molecule. This is probably because of this. In addition, in this observation with an atomic force microscope, it was found that the presence of at least about 100 mer can be sufficiently confirmed.
[0048]
  Next, E. coli (Escherichia coli) RecA protein, and also E. coli (as nuclease)Escherichia coliExonuclease I) was prepared. Further, ATP-γS was prepared as a nucleoside triphosphate or an analog thereof, and a buffer consisting of magnesium acetate and tris acetate was prepared.
[0049]
  Next, in the DNA-protein complex formation step, target DNA, probe DNA, E. coli (Escherichia coli) RecA protein and E. coli (Escherichia
coli) Exonuclease I (Exonuclease I) in the target DNA, the probe DNA, at least E. coli (at least one of the target DNAs) is included in the terminal inclusion region including the 5 ′ end of one of the DNA strands.Escherichia coliTo form a DNA-protein complex (see FIG. 1). Specifically, 1 pmol of probe DNA, 3.0 μg of RecA protein, 4 units of Exonuclease I, 4.8 mM ATP-γS, 600 ng of target DNA in 20 mM magnesium acetate, 30 mM Tris acetate (pH 7.2) And kept at 37 ° C. for 30 minutes. In this reaction, first, the RecA protein binds to the probe DNA to form a probe DNA-RecA protein complex. Subsequently, this probe DNA-RecA protein complex binds to the target DNA to form a DNA-protein complex. In this case, in this DNA-protein complex, it is considered that the probe DNA binds to a region (terminal region) consisting of a base sequence complementary to the probe DNA in the target DNA with at least the RecA protein involved. Exonuclease I is considered to act on the stabilization of this DNA-protein complex. Such a DNA-protein complex is relatively stable.
[0050]
  Next, in the protein deactivation step, the recombinant protein and the nuclease are deactivated to form a DNA complex in which the probe DNA is bound to the inclusion region near the end of the target DNA (see FIG. 1). Specifically, 0.5% (W / Vol) SDS and 0.7 mg / ml proteinase K were added to the above reaction solution and incubated at 37 ° C. for 60 minutes to inactivate RecA protein and exonuclease I (removal). Did protein). As a result, a DNA complex is formed by binding the probe DNA to the inclusion region near the end of the target DNA. Such a DNA complex does not require a special protein or the like in order to maintain its structure, and can maintain a stable state even when a little heat is applied.
[0051]
  Here, an experiment for confirming the formation of the DNA complex was performed. That is, the dye solution was added to the reaction solution after the protein deactivation step, and electrophoresis was performed on a 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide and a photograph of the electrophoresis gel was recorded. The result is shown in lane 5 of FIG. In this lane 5, each of the above reactions was performed using a 600-mer third probe DNA as the probe DNA.
[0052]
  Looking at lane 5 in FIG. 6, three signals were detected as indicated by arrows A, B, and C in the figure. Of these, the signal at the position indicated by arrow A is considered to be derived from the DNA complex due to its size. On the other hand, the signal at the position indicated by arrow B is considered to be derived from the target DNA that does not form a DNA complex because of its size. Further, the signal at the position indicated by the arrow C is considered to be derived from the probe DNA that does not form a DNA complex because of its size.
[0053]
  Next, a comparative experiment whose result is shown in FIG. 6 will be described.
  Lane 1 shows the result of performing the same reaction as Lane 5 except that RecA protein was not added in the above-described DNA-protein complex formation step.
  Lane 2 shows the result of performing the same reaction as Lane 5 except that ATP-γS was not added in the DNA-protein complex formation step described above.
  Lane 3 shows the result of performing the same reaction as Lane 5 except that RecA protein and ATP-γS were not added in the above-described DNA-protein complex formation step.
  Lane 4 shows the result of performing the same reaction as in Lane 5 except that the reaction time (30 minutes in Lane 5) of the above-described DNA-protein complex formation step is 0 minute.
  Lane 6 shows the result of performing the same reaction as Lane 5 except that the reaction time (30 minutes in Lane 5) of the above-described DNA-protein complex formation step is 60 minutes.
[0054]
  As is clear from this, in lanes 1 and 3, no signal was detected at the position indicated by arrow A. That is, in Lane 1 and Lane 3, no DNA complex is formed. From this result, it can be seen that RecA protein is required in the DNA-protein complex formation step for the formation of a stable DNA complex. In lane 2, only a very weak signal was detected at the position indicated by arrow A. From this result, it can be seen that for the formation of a stable DNA complex, it is preferable to add ATP-γS in the DNA-protein complex formation step. In lane 4, only a very weak signal was detected at the position indicated by arrow A. From this result, it can be seen that formation of a stable DNA complex requires a certain amount of reaction time in the DNA-protein complex formation step. On the other hand, in Lane 6, since the DNA-protein complex formation step is performed with a sufficiently long reaction time, a strong signal is detected at the position indicated by arrow A as in Lane 5. That is, a stable DNA complex is formed.
[0055]
  For the other probe DNAs, the above-described reactions were performed, and electrophoresis for confirming the formation of a DNA complex was performed in the same manner as in lane 5 of FIG. The result is shown in FIG.
  Lane 1 shows the result of performing the same reaction as in Lane 5 of FIG. 6 (reaction time of the DNA-protein complex formation step is 30 minutes) using the first probe DNA (150 mer).
  Lane 2 shows the result of performing the same reaction as in Lane 5 of FIG. 6 (reaction time of the DNA-protein complex formation step is 30 minutes) using the second probe DNA (300 mer).
  Lane 3 shows the result of performing the same reaction as in Lane 5 of FIG. 6 (reaction time of the DNA-protein complex formation step is 30 minutes) using the fourth probe DNA (900 mer).
  Lane 4 shows the result of performing the same reaction as in Lane 5 of FIG. 6 (reaction time of the DNA-protein complex formation step is 30 minutes) using the fifth probe DNA (1200 mer).
  Lane 5 shows the result of performing the same reaction as in Lane 6 of FIG. 6 (the reaction time of the DNA-protein complex formation step is 60 minutes) using the first probe DNA (150 mer).
  Lane 6 shows the result of performing the same reaction as in Lane 6 of FIG. 6 (the reaction time of the DNA-protein complex formation step is 60 minutes) using the second probe DNA (300 mer).
  Lane 7 shows the result of performing the same reaction as in Lane 6 of FIG. 6 (the reaction time of the DNA-protein complex formation step is 60 minutes) using the fourth probe DNA (900 mer).
  Lane 8 shows the result of performing the same reaction as in Lane 6 of FIG. 6 (the reaction time of the DNA-protein complex formation step is 60 minutes) using the fifth probe DNA (1200 mer).
[0056]
  In any of the lanes in FIG. 7, signals were observed at positions larger (upper in the figure) and smaller (lower in the figure) than the signals indicated by arrows in the figure. The signal at a large position is considered to be derived from each DNA complex because of its size. On the other hand, the signal at the position indicated by the arrow is considered to be derived from the target DNA not forming a DNA complex due to its size. Further, the signal at a small position is considered to be derived from each probe DNA that does not form a DNA complex because of its size. Thus, it can be seen that a DNA complex is formed using any probe DNA.
[0057]
  Next, the DNA complex observed in the gel photographs of FIGS. 6 and 7 was cut out from the gel and purified. Purification was performed using a Gel extraction kit (Amersham Pharmacia) according to the protocol. Thereafter, a dye solution was added to each purified DNA solution, and electrophoresis was performed on a 1% agarose gel. After the electrophoresis, the agarose gel was stained with ethidium bromide, and a photograph of the electrophoresis gel was recorded. The result is shown in FIG.
  Lane 1 shows the result of cutting out and purifying the DNA complex of Lane 1 in FIG. 7, that is, the result of electrophoresis of the DNA complex formed using the first probe DNA.
  Lane 2 shows the result of cutting out and purifying the DNA complex of Lane 2 in FIG. 7, that is, the result of electrophoresis of the DNA complex formed using the second probe DNA.
  Lane 3 shows the result of cutting out and purifying the DNA complex of Lane 5 in FIG. 6, that is, the result of electrophoresis of the DNA complex formed using the third probe DNA.
  Lane 4 shows the result of cutting out and purifying the DNA complex of Lane 3 in FIG. 7, that is, the result of electrophoresis of the DNA complex formed using the fourth probe DNA.
  Lane 5 shows the result of cutting out and purifying the DNA complex of Lane 4 in FIG. 7, that is, the result of electrophoresis of the DNA complex formed using the fifth probe DNA.
[0058]
  In any lane in FIG. 8, signals derived from the respective DNA complexes were observed, and few other signals were observed. From this result, it can be seen that the DNA complex was separated and purified with high purity in any sample.
[0059]
  Next, in the AFM detection step, the purified DNA complex was observed with an atomic force microscope. The procedure for AFM specimen preparation was as follows. Magnesium chloride was added to the DNA solution to a final concentration of 10 mM, and 50 μl was dropped on a fresh surface immediately after cleaving of a 10 mm square mica substrate and allowed to stand at room temperature for 5 minutes. Next, this was washed with 5 ml of sterilized water, blown away with nitrogen gas, and further dried under vacuum at 40 ° C. for 15 minutes. Gene visualization conditions were measured in the tapping mode using a silicon single crystal cantilever (D-NCH-10V) with the sample prepared above set on a scanning probe microscope (Nanoscope IIIa) (Degital Instruments). did. The scan range was 0.5-2 μm in the XY direction and 2 nm in the height (Z-axis) direction. DNA length was measured using NIHimage, a freeware.
[0060]
  The result of using the first probe DNA (150mer) as the probe DNA is shown in FIG. 9, and the photograph schematically shown in FIG. 9 is shown in FIG. FIG. 11 shows the result of using the second probe DNA (300 mer) as the probe DNA, and FIG. 12 schematically shows the photograph of FIG. FIG. 13 shows the result of using the third probe DNA (600mer) as the probe DNA, and FIG. 14 schematically shows the photograph of FIG. Moreover, the result of using the fourth probe DNA (900 mer) as the probe DNA is shown in FIG. 15, and the photograph schematically shown in FIG. 15 is shown in FIG. Moreover, the result of using the fifth probe DNA (1200 mer) as the probe DNA is shown in FIG. 17, and the photograph schematically shown in FIG. 17 is shown in FIG.
[0061]
  As shown in FIGS. 9 and 10, the DNA complex of the target DNA and the first probe DNA could be observed with an atomic force microscope. In this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA. A part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound.
  Moreover, as shown in FIGS. 11 and 12, the DNA complex of the target DNA and the second probe DNA could be observed with an atomic force microscope. Also in this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA. A part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound.
  Moreover, as shown in FIGS. 13 and 14, the DNA complex of the target DNA and the third probe DNA could be observed with an atomic force microscope. Also in this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA. A part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound.
  Further, as shown in FIGS. 15 and 16, the DNA complex of the target DNA and the fourth probe DNA could be observed with an atomic force microscope. Also in this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA. A part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound.
  Moreover, as shown in FIGS. 17 and 18, the DNA complex of the target DNA and the fifth probe DNA could be observed with an atomic force microscope. Also in this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA. A part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound.
[0062]
  If the DNA complex is detected as described above, the DNA complex can be visualized and confirmed, so that the structure and form of the DNA complex can also be examined. Moreover, since each DNA complex is visualized, the DNA complex can be reliably detected even if the amount of the sample is small. Furthermore, the time required for detection is reduced compared to the methods using Southern hybridization, since there is no need to label a part of the DNA, and it is not necessary to perform electrophoresis of the DNA complex or to dry the gel. Can be shorter.
[0063]
  Various recombinant proteins can be appropriately selected as the recombinant protein used in the DNA-protein complex formation step. However, considering the availability, safety, and functionality, they are derived from E. coli as in this embodiment. It is preferred to use RecA protein.
  In addition, in the DNA-protein complex formation step, by adding ATP-γS and reacting, a DNA protein complex can be formed more efficiently, and as a result, a DNA complex can be formed more efficiently.
  In addition, single-stranded DNA can bind to double-stranded DNA to form a DNA complex even if its chain length is short. However, if the single-stranded DNA is short, specifically, shorter than 60 mer, it is difficult to distinguish the double-stranded DNA from the DNA complex when observed with an atomic force microscope. That is, it becomes more difficult to detect the DNA complex. Therefore, when a single-stranded DNA having a base sequence of 60 mer or more is used as in this embodiment, the DNA complex can be detected more reliably when observed with an atomic force microscope.
[0064]
(Embodiment 2)
  Next, a second embodiment will be described. Note that the description of the same parts as those in the first embodiment is omitted or simplified.
  In the present embodiment, a nuclease treatment step for reacting the DNA complex with a nuclease is performed after the protein deactivation step and before the AFM detection step. The rest is basically the same as the first embodiment.
[0065]
  More specifically, as in the first embodiment, pSP64poly (A) DNA is cleaved into three DNA fragments with the restriction enzyme RsaI as the target DNA (double-stranded DNA), and the largest DNA fragment ( About 1.9 kbp) was purified. In addition, as a probe DNA (single-stranded DNA), a 150-mer first probe DNA used in the first embodiment was prepared. In addition, as in Embodiment 1, the recombinant protein is Escherichia coli (Escherichia coli) RecA protein as a nuclease, E. coli (Escherichia coliExonuclease I) was prepared. Further, ATP-γS was prepared as a nucleoside triphosphate or an analog thereof, and a buffer consisting of magnesium acetate and tris acetate was prepared.
[0066]
  Then, in the DNA-protein complex formation step, the target DNA, probe DNA, E. coli (Escherichia coli) RecA protein and E. coli (Escherichia coli) Exonuclease I (Exonuclease I) in the target DNA, the probe DNA, at least E. coli (at least one of the target DNAs) is included in the terminal inclusion region including the 5 ′ end of one of the DNA strands.Escherichia coli) To form a DNA-protein complex formed by binding in a state involving the RecA protein.
  Next, in the protein deactivation step, the recombinant protein and the nuclease were deactivated in the same manner as in the first embodiment to form a DNA complex in which the probe DNA was bound to the inclusion region near the end of the target DNA. .
  Thereafter, in the same manner as in Embodiment 1, the reaction solution was electrophoresed, and then the DNA conjugate was cut out from the gel and purified.
[0067]
  Next, in this embodiment, a nuclease treatment step for reacting the DNA complex with a nuclease was performed. Specifically, for the purified DNA solution, 10 units of E. coli (Escherichia coli) Exonuclease I (manufactured by Epicentre), 33 mM Tris-Acetate (pH 7.8), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT at 37 ° C. for 20 minutes.
[0068]
  Next, in the AFM detection step, the DNA complex was observed with an atomic force microscope in the same manner as in the first embodiment. The result is shown in FIG. Moreover, what showed the photograph of FIG. 19 typically is shown in FIG.
  As shown in FIGS. 19 and 20, the DNA complex of the target DNA and the first probe DNA could be observed with an atomic force microscope. In the figure, two DNA complexes are observed. In this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA, as in the first embodiment. In the first embodiment, as shown in FIG. 9 and FIG. 10, a part of the target DNA (part to which the probe DNA is bound) is in a single-stranded state as the double-stranded DNA is unwound (beard). Free). In contrast, in this embodiment, about 100 DNA complexes were observed, but no DNA complex having a whisker-like portion was observed as shown in FIGS. The observed length of the DNA complex was the same as the length of the target DNA. From this, it is considered that a part of the target DNA liberated in a whisker shape is not degraded by a nuclease specific to single-stranded DNA in the nuclease treatment step.
[0069]
  As described above, also in this embodiment, since the DNA complex can be visualized and confirmed, the structure and form of the DNA complex can be examined, and the same effects as in the first embodiment can be obtained. Can do. In the present embodiment, since the nuclease treatment step is performed after the protein deactivation step and before the AFM detection step, a DNA complex having no DNA strands that are beard-like can be observed by AFM.
[0070]
(Embodiment 3)
  Next, a third embodiment will be described. Note that description of the same parts as those in the first embodiment or the second embodiment is omitted or simplified.
  In this embodiment, the point which makes it react using ATP instead of ATP-gammaS in a DNA-protein complex formation process differs from the said Embodiment 1. FIG. The rest is basically the same as in the first embodiment.
[0071]
  More specifically, as in the first embodiment, pSP64poly (A) DNA is cleaved into three DNA fragments with the restriction enzyme RsaI as the target DNA (double-stranded DNA), and the largest DNA fragment ( About 1.9 kbp) was purified. In addition, as a probe DNA (single-stranded DNA), a 900-mer fourth probe DNA also used in the first embodiment was prepared. In addition, as in Embodiment 1, the recombinant protein is Escherichia coli (Escherichia coli) RecA protein as a nuclease, E. coli (Escherichia coliExonuclease I) was prepared. Moreover, what consists of magnesium acetate and tris acetate was prepared as a buffer solution. As described above, in this embodiment, ATP is prepared as nucleoside triphosphate or an analog thereof.
[0072]
  Then, in the DNA-protein complex formation step, the target DNA, probe DNA, E. coli (Escherichia coli) RecA protein and E. coli (Escherichia coli) Exonuclease I (Exonuclease I) in the target DNA, the probe DNA, at least E. coli (at least one of the target DNAs) is included in the terminal inclusion region including the 5 ′ end of one of the DNA strands.Escherichia coli) To form a DNA-protein complex formed by binding in a state involving the RecA protein. That is, except that ATP was used instead of ATP-γS, the same reaction as in Embodiment 1 was performed to form a DNA-protein complex.
[0073]
  Next, in the AFM detection step, the DNA complex was observed with an atomic force microscope in the same manner as in the first embodiment. The result is shown in FIG. FIG. 22 schematically shows the photograph of FIG.
  As shown in FIGS. 21 and 22, the DNA complex of the target DNA and the fourth probe DNA could be observed with an atomic force microscope. In the figure, two DNA complexes are observed. In this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA, as in the first embodiment. In the first embodiment, as shown in FIGS. 15 and 16, a part of the target DNA (portion where the probe DNA is bound) is in a single-stranded state by unwinding the double-stranded DNA (beard). Free). In contrast, in this embodiment, about 100 DNA complexes were observed, but as shown in FIGS. 21 and 22, no DNA complex having a whisker-like portion was observed. The observed length of the DNA complex was the same as the length of the target DNA. That is, the same result as in the second embodiment was obtained. The reason for this is that during the DNA-protein complex formation process, ATP was biochemically degraded, the RecA protein was removed from the single-stranded DNA that was released in a whisker-like manner, and the single-stranded DNA was degraded by a nuclease. It is thought that it was gone.
[0074]
  As described above, also in this embodiment, since the DNA complex can be visualized and confirmed, the structure and form of the DNA complex can be examined, and the same effects as in the first or second embodiment can be obtained. Obtainable. In the present embodiment, since ATP is used instead of ATP-γS in the DNA-protein complex formation step, a DNA complex having no DNA strands that are beard-like can be observed by AFM.
[0075]
(Embodiment 4)
  Next, a fourth embodiment will be described. In addition, description of the part similar to either of the said Embodiment 1-Embodiment 3 is abbreviate | omitted or simplified.
  In this embodiment, the point which makes it react using GTP instead of ATP-gammaS in a DNA-protein complex formation process differs from the said Embodiment 1. FIG. The rest is basically the same as in the first embodiment.
[0076]
  More specifically, as in the first embodiment, pSP64poly (A) DNA is cleaved into three DNA fragments with the restriction enzyme RsaI as the target DNA (double-stranded DNA), and the largest DNA fragment ( About 1.9 kbp) was purified. In addition, as a probe DNA (single-stranded DNA), a 900-mer fourth probe DNA also used in the first embodiment was prepared. In addition, as in Embodiment 1, the recombinant protein is Escherichia coli (Escherichia coli) RecA protein as a nuclease, E. coli (Escherichia coliExonuclease I) was prepared. Moreover, what consists of magnesium acetate and tris acetate was prepared as a buffer solution. As described above, in this embodiment, GTP is prepared as nucleoside triphosphate or an analog thereof.
[0077]
  Then, in the DNA-protein complex formation step, the target DNA, probe DNA, E. coli (Escherichia coli) RecA protein and E. coli (Escherichia coli) Exonuclease I (Exonuclease I) in the target DNA, the probe DNA, at least E. coli (at least one of the target DNAs) is included in the terminal inclusion region including the 5 ′ end of one of the DNA strands.Escherichia coli) To form a DNA-protein complex formed by binding in a state involving the RecA protein. That is, except that GTP was used in place of ATP-γS, a reaction similar to that in Embodiment 1 was performed to form a DNA-protein complex.
[0078]
  Next, in the AFM detection step, the DNA complex was observed with an atomic force microscope in the same manner as in the first embodiment. The result is shown in FIG. FIG. 24 schematically shows the photograph of FIG.
  As shown in FIGS. 23 and 24, the DNA complex of the target DNA and the fourth probe DNA could be observed with an atomic force microscope. In the figure, two DNA complexes are observed. In this DNA complex, the probe DNA is bound to a region complementary to the probe DNA in the target DNA, as in the first embodiment. In the first embodiment, as shown in FIGS. 15 and 16, a part of the target DNA (portion where the probe DNA is bound) is in a single-stranded state by unwinding the double-stranded DNA (beard). Free). In contrast, in the present embodiment, about 100 DNA complexes were observed, but as shown in FIGS. 23 and 24, no DNA complex having a whisker-like portion was observed. The observed length of the DNA complex was the same as the length of the target DNA. That is, the same results as those in Embodiments 2 and 3 were obtained. The reason for this is that, as in the case of Embodiment 3 above, during the DNA-protein complex formation step, GTP is biochemically degraded, and RecA protein is released from the single-stranded DNA released in a whisker-like manner, It is thought that the single-stranded DNA is no longer degraded by nuclease.
[0079]
  As described above, also in this embodiment, since the DNA complex can be visualized and confirmed, the structure and form of the DNA complex can be examined, and the same effects as in the first to third embodiments are obtained. Obtainable. Moreover, in this embodiment, since GTP is used instead of ATP-γS in the DNA-protein complex formation step, a DNA complex without a DNA strand that is released in a beard shape can be observed by AFM.
[0080]
  In the above, the present invention has been described with reference to the embodiments. However, the present invention is not limited to the above embodiments, and it is needless to say that the present invention can be appropriately modified and applied without departing from the gist thereof.
[0081]
【The invention's effect】
  According to the present invention, a DNA complex in which double-stranded DNA and single-stranded DNA are bound can be visualized and detected.
[0082]
[Sequence Listing]
<110> Aisin Cosmos Research Institute
         AISIN COSMOS R & D CO., LTD
         Kazusa DNA Research Institute
         Kazusa DNA Research Institute
<120> Nucleic acid detection method
<130> 2002 AICO01
<160> 6
<210> 1
<211> 150
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 1
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 150
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 2
gccgggaagc tagagtaagt agtt 24
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 3
actcaccagt cacagaaaag catc 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 4
gttttaaatc aatctaaagt atat 24
<210> 5
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 5
ggtggcctaa ctacggctac acta
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthesis with reference to the base sequence of pSP64poly (A) + DNA
<400> 6
tcgtgcgctc tcctgttccg accc 24
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing the formation of a DNA complex with respect to Embodiment 1.
FIG. 2 is a photograph replacing a drawing showing the result of electrophoresis of a gel-filtered target DNA in the first embodiment.
FIG. 3 is a photograph instead of a drawing showing the results of electrophoresis of PCR products for preparing the second to fifth probe DNAs in the first embodiment.
FIG. 4 is a photograph replacing a drawing showing a state where the target DNA and the third probe DNA are observed with an atomic force microscope in the first embodiment.
FIG. 5 is an explanatory diagram schematically showing the photograph of FIG. 4 regarding the first embodiment.
FIG. 6 is a photograph replacing a drawing, showing the results of electrophoresis of DNA complexes formed under various reaction conditions in the first embodiment.
FIG. 7 is a photograph, instead of a drawing, showing the results of electrophoresis of DNA complexes formed with various probe DNAs in the first embodiment.
FIG. 8 is a photograph replacing a drawing, showing the results of electrophoresis of various purified DNA complexes in the first embodiment.
FIG. 9 is a photograph replacing a drawing, showing a state where a DNA complex formed using the first probe DNA is observed with an atomic force microscope in the first embodiment.
FIG. 10 is an explanatory diagram schematically showing the photograph of FIG. 9 regarding the first embodiment.
FIG. 11 is a photograph replacing a drawing showing a state where a DNA complex formed using the second probe DNA is observed with an atomic force microscope in the first embodiment.
FIG. 12 is an explanatory diagram schematically showing the photograph of FIG. 11 regarding the first embodiment.
FIG. 13 is a photograph replacing a drawing, showing a state where a DNA complex formed using the third probe DNA is observed with an atomic force microscope in the first embodiment.
FIG. 14 is an explanatory diagram schematically showing the photograph of FIG. 13 regarding the first embodiment.
FIG. 15 is a photograph replacing a drawing, showing a state where a DNA complex formed using the fourth probe DNA is observed with an atomic force microscope in the first embodiment.
FIG. 16 is an explanatory diagram schematically showing the photograph of FIG. 15 regarding the first embodiment.
FIG. 17 is a photograph replacing a drawing, showing a state where a DNA complex formed using the fifth probe DNA is observed with an atomic force microscope in the first embodiment.
FIG. 18 is an explanatory diagram schematically showing the photograph of FIG. 17 regarding the first embodiment.
FIG. 19 is a photograph replacing a drawing, showing a state where a DNA complex is observed with an atomic force microscope in the second embodiment.
FIG. 20 is an explanatory diagram schematically showing the photograph of FIG. 19 regarding the second embodiment.
FIG. 21 is a photograph replacing a drawing, showing a state where a DNA complex is observed with an atomic force microscope in the third embodiment.
FIG. 22 is an explanatory diagram schematically showing the photograph of FIG. 21 with respect to the third embodiment.
FIG. 23 is a photograph replacing a drawing, showing a state where a DNA complex is observed with an atomic force microscope in the fourth embodiment.
FIG. 24 is an explanatory diagram schematically showing the photograph of FIG. 23 with respect to the third embodiment.

Claims (3)

Linear double-stranded DNA,
A nucleotide sequence starting from the 5 'end near the base of one of the DNA strands of the double stranded DNA, linear single-stranded DNA including a phase complementary to the nucleotide sequence,
A recombinant protein that is a RecA protein of Escherichia coli ,
And
Escherichia coli exonuclease I nuclease,
In the double-stranded DNA, a complementary region having the complementary base sequence in the single-stranded DNA in a region near the end including the vicinity of the 5 ′ end of the one DNA strand. A DNA-protein complex forming step of forming a DNA-protein complex formed by binding in a state where at least the recombinant protein is involved,
A protein inactivation step of inactivating the recombinant protein and the nuclease to form a DNA complex in which the complementary region of the single-stranded DNA is bound to the inclusion region near the end of the double-stranded DNA;
An AFM detection step of detecting the DNA complex by an atomic force microscope ,
In the DNA-protein complex formation step, ATP-γS is added,
A nucleic acid detection method comprising a nuclease treatment step of reacting the DNA complex with the nuclease after the protein deactivation step and before the AFM detection step . The nucleic acid detection method according to claim 1 ,
A nucleic acid detection method comprising adding a nucleoside triphosphate in the DNA-protein complex formation step. The nucleic acid detection method according to claim 1 or 2 ,
The nucleic acid detection method, wherein the single-stranded DNA comprises a base sequence of 60 mer or more.

Images