JP4514482B2 - Detection method of target nucleotide sequence - Google Patents

Description

  The present invention relates to a method for detecting whether or not a target base sequence exists in sample DNA or RNA by utilizing a binding action by hybridization between sample DNA or RNA and probe DNA or RNA. The substance to be handled can be considered in the same manner whether it is DNA or RNA. The case where the sample is DNA and the probe is DNA is described below.

Conventionally, as a method for detecting whether a target base sequence exists in a sample DNA, a DNA having a base sequence complementary to the target base sequence to be detected is prepared as a probe DNA and fixed to glass or a membrane. To make a DNA chip, hybridize the sample DNA and the probe DNA immobilized on the DNA chip, and if the sample DNA has the target base sequence, the site having the target base sequence in the sample DNA and the DNA chip The probe DNA immobilized on is hybridized and bound. When there is no target base sequence, it will not be bound to the probe DNA, so it will be floating in the solution. After the hybridization, when the DNA chip is washed, the floating DNA is washed away, and the immobilized DNA is not washed away. Therefore, since the sample DNA hybridized with the probe DNA is bound, it will not be washed away even if the DNA chip is washed. The method for detecting the probe DNA that is hybridized is to preliminarily label the sample DNA with a fluorescent label, excite it with a light source such as a lamp or laser, emit light, read the image with a DNA chip reader, Whether DNA is bound or not is detected by the one-step detection performed by directly hybridizing the probe DNA with the sample DNA using the action of hybridization to detect the presence or absence of the target base sequence (see Non-Patent Document 1).
FIG. 2 shows an example of a method for detecting a target base sequence using a DNA chip that is widely used recently. This example detects whether four different target base sequences (1), target base sequence (2) 2, target base sequence (3) 3, target base sequence (4) 4 are present in sample DNA 5 It shows the technique to do. In this example, the sample DNA having the target base sequence (1) 1 and the target base sequence (4) 4 is detected as an example. FIG. 2 (A) shows sample DNA 5 preliminarily labeled with fluorescence, which is sample DNA 5 in which the target base sequence (1) 1 and target base sequence (4) 4 are present. FIG. 2 (B) shows a DNA group 14 in a pair of target base sequence (1) 1 to target base sequence (4) 4 to be detected. DNA 1a of target base sequence (1) and target base sequence (1) Complementary strand DNA 1b, DNA 2a of target base sequence (2) and complementary strand DNA 2b of target base sequence (2), DNA 3a of target base sequence (3), complementary strand DNA 3b of target base sequence (3) and target base sequence (4) DNA 4a and the complementary strand DNA 4b of the target base sequence (4). At this time, since the relationship between the DNA 1a of the target base sequence (1) and the complementary strand DNA 1b of the target base sequence (1) may be hybridized, the complementary relationship may or may not be perfect match. In FIG. 2C, the complementary strand DNAs 1b to 4b in FIG. 2B are immobilized on the glass or membrane 13 as the probe DNA group 8, and the DNA chip 9 in FIG. In FIG. 2E, the sample DNA 5 and the probe DNA group 8 fixed to the DNA chip 9 are put into the hybridization solution 10 and hybridized. At that time, since the site of the target base sequence (1) 1 of the sample DNA 5 and the complementary strand DNA 1b of the target base sequence (1) are in a complementary chain relationship, the site of the target base sequence (1) 1 of the sample DNA 5 and the target base The complementary strand DNA 1b of sequence (1) is hybridized and bound. FIG. 2F shows the reading of the DNA chip 9 after hybridization, and the method is that the fluorescent label 6 is excited by the lamp 11 to emit light and is read by the DNA chip reader 7. In this example, the position of the complementary strand DNA 1b of the target base sequence (1) 1 that is hybridized with the site of the target base sequence (1) 1 of the sample DNA 5 is illuminated. FIG. 2G shows an image 12 read by the DNA chip reader 7 and it can be seen that the target base sequence (1) 1 exists in the sample DNA 5 from the shining position. At present, it is detected in this way whether the target base sequence is present in the sample DNA.
“Science” Vol.270, p467-470

  In the conventional method, when the amount of sample DNA is small, probe DNA is immobilized on glass or a membrane, so that only one sample DNA can be hybridized to one probe DNA. For this reason, when there are a plurality of types of target base sequences in the sample DNA, only one can be bound, so that there is a problem that other target base sequences cannot be detected. For this reason, a method of amplifying and increasing the sample DNA is employed, but there is a problem that the conditions for amplification are difficult, such as the sample DNA being unknown and the molecular size being different. Further, in the hybridization using a DNA chip, there is a problem that the sample DNA encounters an immobilized probe DNA because the space for hybridization is too large. Furthermore, since the molecular weights of the sample DNA and the probe DNA are greatly different, there is a problem that it is difficult to set experimental conditions such as a temperature as a hybridization condition.

  The present invention has been made in view of such problems of the prior art, and allows a plurality of probe DNAs to be hybridized to a sample DNA, so that even if a plurality of target base sequences exist in the sample DNA, the present invention can be easily performed. It is an object of the present invention to provide a method capable of being detected.

The present invention is a method for detecting a target base sequence in sample DNA or RNA, comprising the following steps:
(1) Contacting sample DNA or RNA with probe DNA or RNA in a liquid to form a hybridization complex;
(2) isolating the hybridization complex;
(3) dissociating the complex and separating the probe DNA or RNA;
(4) Identify the probe DNA or RNA and detect the target base sequence in the sample DNA or RNA;
Relates to a method comprising:

In the present invention, i) when the sample is DNA and the probe is DNA, ii) when the sample is RNA and the probe is RNA, iii) when the sample is DNA and the probe is RNA, iv) when the sample is RNA and the probe is DNA There are four aspects of the case. It can be similarly considered whether the substance to be handled is DNA or RNA. The case where the sample is DNA and the probe is DNA is described below.
In the present invention, a probe DNA that has been conventionally immobilized on a glass or membrane, which is conventionally called a DNA chip, is not immobilized, but an aqueous solution of sample DNA and probe DNA is prepared. To form a hybridization complex. This makes it possible to hybridize a plurality of types of probe DNA to the sample DNA even when the sample DNA is small. Thereafter, by isolating the hybridization complex, separating the probe DNA from the hybridization complex, and identifying the probe, the target base sequence present in the sample DNA can be detected.

  That is, by hybridizing the sample DNA and the probe DNA in one tube, the space for hybridization can be narrowed, and the probability of encountering both can be increased. Furthermore, by performing hybridization in one tube, it becomes possible to detect even if the amount of sample DNA is small. In addition, a plurality of probe DNAs can be used, and a plurality of probe DNAs can be hybridized to one sample DNA.

The isolated probe DNA can be identified by preparing a c-probe DNA that is complementary to the probe DNA and using hybridization. That is, c-probe DNA having a complementary strand relationship with probe DNA is immobilized on glass or a membrane, prepared in advance as a c-probe DNA chip, hybridized with separated probe DNA, and identified. At this time, since the probe DNA and the c-probe DNA on the c-DNA chip have substantially the same molecular weight, they can be stably hybridized and bound under easy conditions. When the probe DNA is fluorescently labeled and the c-DNA chip is read with a DNA chip reader, the place where the selected and extracted probe DNA is bound to the c-probe DNA shines. From the position on the c-DNA chip, It is possible to specify what probe DNA it is. That is, it can be seen that the base sequence of the c-probe DNA bound to the probe DNA is the target base sequence present in the sample DNA.
In short, in the present invention, a single sample DNA is hybridized with one probe DNA. In the present invention, a plurality of probe DNAs can be hybridized with one sample DNA. It is characterized by detecting with c-probe DNA of complementary strand probe DNA. When reading the c-DNA chip with light, it is necessary to attach a fluorescent label to the probe DNA before the hybridization between the extracted probe DNA and the c-probe DNA. Fluorescent labeling of probe DNA (Nucleic Acids Research Vol. 13, pages 2399-2412, DNA Sequence Vol. 4, vol. 135-141) is already well known to those skilled in the art, and commercially available DNA synthesis Oligonucleotides to which various fluorescent substances are added can be synthesized using a machine. In addition, contract synthesis of fluorescently labeled oligonucleotides by companies has been put into practical use.

  Multiple types of base sequences can be detected with a small amount of DNA sample.

Hereinafter, an embodiment of the present invention will be described. FIG. 1 is a diagram for explaining the principle of the present invention, and shows a search means for determining whether or not a target base sequence exists in sample DNA. In this example, there are four types of target base sequences to be obtained, and the sample DNA 5 uses the example in which the target base sequence (1) 1 and target base sequence (4) 4 are present to represent the search method. ing. FIG. 1A shows a sample DNA 5 to be searched for a target base sequence. The target base sequence (1) 1 and the target base sequence (4) 4 are present, and magnetic beads 17 for controlling the sample DNA 5 are bound. Yes. FIG. 1B shows a pair of DNA groups 14 for searching for a target base sequence. DNA 1a of the target base sequence (1), complementary strand DNA 1b of the target base sequence (1), target base sequence ( 2) DNA 2a and complementary strand DNA 2b of target base sequence (2), DNA 3a of target base sequence (3) and complementary strand DNA 3b of target base sequence (3), DNA 4a of target base sequence (4) and target base sequence (4 ) Complementary strand DNA 4b. FIG. 1C shows a probe DNA group 8 for hybridization with a sample DNA for searching. DNA 1b of target base sequence (1) 1, DNA 2b of target base sequence (2) 2, target base sequence (3 3) DNA 3b of 3 and DNA 4b of the desired base sequence (4) 4. FIG. 1 (D) is a means for detecting the probe DNA hybridized with the sample DNA 5, from the complementary strand DNA 1a of the desired base sequence (1) in the complementary strand relationship of the probe DNA group 8 to the desired base sequence (4). The c-probe DNA group 15 of the complementary strand DNA 4a is immobilized on glass or a membrane 13 to form a c-DNA chip 16. In FIG. 1E, sample DNA 5 and probe DNA group 8 are placed in a hybridization solution 10 and hybridized with one tube 19. At this time, if the sample DNA 5 has a base sequence that is homologous to the probe DNA, it is hybridized and bound, and the unbound probe DNA floats in the solution. In this example, the complementary strand DNA 1b of the target base sequence (1) and the complementary strand DNA 4b of the target base sequence (4) bind to the sites of the target base sequence (1) 1 and the target base sequence (4) 4 of the sample DNA 5. become.
1 (F) and FIG. 1 (G) are means for separating the probe DNA bound to the sample DNA 5 and the unbound probe DNA after the hybridization of FIG. 1 (E). Fix as shown in FIG. 1 (F) and transfer the solution from FIG. 1 (F) to FIG. 1 ( G ). At this time, the complementary strand DNA 1b of the target base sequence (1) of the probe DNA bound to the sample DNA 5 and the complementary strand DNA 4b of the target base sequence (4) do not move, and are unbound probes floating in the solution. The complementary strand DNA 2b of the target base sequence (2) of DNA and the complementary strand DNA 3b of the target base sequence (3) move as shown in FIG. 1 (G). By doing so, the bound probe DNA and the unbound probe DNA can be separated, and the probe DNA bound to the sample DNA 5 can be selected.
In FIG. 1 (H), in order to separate the bound probe DNA, the complementary strand DNA 1b of the target base sequence (1) and the complementary strand DNA 4b of the target base sequence (4) are separated from the sample DNA 5. Separation takes place under alkaline conditions or with heat.
FIGS. 1 (I) and 1 (J) separate sample DNA 5 and probe DNA that have been separated, and sample DNA 5 is moved from FIG. 1 (H) to FIG. 1 (I). As a result, the probe DNA bound in FIG. 1 (J) remains, and the sample DNA 5 and the complementary strand DNA 1b of the target base sequence (1) of the probe DNA and the complementary strand DNA 4b of the target base sequence (4) are separated. be able to. After that, for reading, the fluorescent substance is labeled on the probe DNA separated and extracted as shown in FIG. 1 (J), but the timing of labeling the fluorescent substance may be performed at the time of FIG. 1 (C).
In FIG. 1 (K), in order to detect the type of probe DNA separated and extracted, the separated probe DNA of FIG. 1 (J) and the c-DNA chip 16 of FIG. It is hybridized with the c-probe DNA group 15 in a complementary strand relationship with the probe DNA. As a result, the DNA 1a of the target base sequence (1) and the complementary strand DNA 1b of the target base sequence (1) and the DNA 4a of the target base sequence (4) and the complementary strand DNA 4b of the target base sequence (4) are hybridized and combined. The
FIG. 1 (L) shows detection of a hybridized probe DNA, and a c-DNA chip is read by a conventional DNA chip reader 7.
FIG. 1 (M) is an image obtained by reading a c-DNA chip. Since the position where the extracted probe DNA is bonded is lit, it can be determined which probe DNA is bonded. In this example, it can be seen that the probe DNAs of the target base sequence (1) and the target base sequence (4) were bound to the sample DNA.
Thus, by obtaining the probe DNA bound to the c-DNA chip, it is possible to detect which target base sequence is present in the sample DNA. Although the method for detecting the base sequence using an automated DNA chip has been described here for the classification and detection of the probe DNA bound to the sample DNA, the method for classifying and detecting the probe DNA bound to the sample DNA is described below. It may be a technique. Of course, the method of detecting the probe DNA of the present invention using a complementary-strand probe DNA can be applied to devices other than DNA chips.
Hereinafter, an example in which the probe selectively bound to the sample DNA is collected by the above method will be described.

Hybridization of sample DNA and probe As the sample DNA for detecting the target nucleotide sequence by the method of the present invention, the DNA shown in SEQ ID NO: 1 was used. As will be described later, biotin was covalently bound to the 5 ′ end for selection after hybridization. As probes having a sequence complementary to the sample DNA, DNA1 and DNA2 shown in SEQ ID NOs: 2 and 3 having sequences complementary to the base sequences 20-35 and 41-55 of the sample DNA, respectively, Synthesized. Further, DNA3, DNA4, and DNA5 shown in SEQ ID NOs: 4 to 6 were used as DNAs having no complementary strand sequence in this sample DNA. All of these DNAs including the sample DNA and the probe were mixed and hybridized. Hybridization conditions were 5 x SSC, 0.5% SDS, 0.2 mg / ml. Activated DNA (calf thymus DNA digested with DNase I) was mixed with the above DNA and heat-treated at 95 ° C for 3 minutes. And then left at 42 ° C. for 10 minutes.

Selective recovery of probe DNA bound to sample DNA Biotin bound to sample DNA was used for the purpose of this experiment. The biotinylation of DNA can be carried out by the method described in Tetrahedron Letters, 32, 1715-1718, Nucleic Acids Research, 20, 6253-6259 (biotin in this experiment). The sample DNA was converted from DNA synthesis commissioned by Sigma Genosys). That is, biotinylated DNA was collected by the following procedure using a streptavidin that specifically binds to biotin immobilized on magnetic beads (MACS Separation system, Miltenyi Biotech). The hybridization solution was mixed with 100 μl of MACS Streptavidin MicroBeads and applied to a column set on a strong permanent magnet. The column was preliminarily flowed with a hybridization solution not containing DNA as an equilibration buffer. Similarly, 100 μl of the hybridization solution was flowed 5 times to the column after the sample was applied, and then 100 μl of TE buffer was further flowed 3 times to wash away non-specifically bound DNA. Thereafter, the column was removed from the magnetic plate, and the sample DNA to which the probe was bound was collected in a tube by flowing TE buffer.

Confirmation of selective recovery of probe for sample DNA To detect the probe bound to the recovered sample DNA, treat the DNA solution finally recovered with TE buffer at 95 ° C by the above method. The hybridized DNA was removed by rapid cooling. The 5 ′ end of the probe DNA was radiolabeled by adding radioactive [γ ³² P] ATP and polynucleotide kinase to the solution. The reaction product was subjected to 20% polyacrylamide gel electrophoresis in the presence of 8 M urea, and a DNA band was detected by autoradiography. Based on the position of the obtained band, the type of DNA bound to the target DNA was estimated based on the DNA chain length. That is, if the target probe selectively binds to the sample DNA and is recovered together with the sample DNA by the magnet method, DNA1 and DNA2 are detected as 15- and 16-strand DNA bands, and the others However, the experimental result was exactly the same, indicating that the target probe having a sequence complementary to the sample DNA was specifically bound. The results are shown in FIG. From this result, it was shown that a plurality of probe DNAs were bound to one kind of sample DNA with the same efficiency, and it was shown that the principle of the present invention can work effectively.

It is a figure explaining the principle of this invention, and shows the search means whether the target base sequence exists in sample DNA. FIG. 2 is a diagram for explaining an example of a method for detecting a target base sequence using a DNA chip that has been frequently used recently. It is an electrophoretic diagram for detecting probe DNA bound to sample DNA. The DNA solution recovered together with the sample DNA by the magnet method was radioactively labeled, and the chain length was detected by denaturing polyacrylamide gel electrophoresis. Lanes 1 and 7 are labeled with the recovered solution. In lanes 2 to 6, the probe DNAs (DNA1 to 5 ) used in this experiment were directly labeled and electrophoresed. Lane 2, DNA1; Lane 3, DNA2; Lane 4, DNA3; Lane 5, DNA4; Lane 6, DNA5.

Explanation of symbols

1 target base sequence (1);
1a DNA of the target base sequence (1);
1b complementary strand DNA of the target base sequence (1);
2 Target nucleotide sequence (2)
2a DNA of the target nucleotide sequence (2)
2b complementary strand DNA of the target base sequence (2);
3 Target base sequence (3);
3a DNA of the target base sequence (3);
3b complementary strand DNA of the target base sequence (3);
4 Target base sequence (4);
4a DNA of the target base sequence (4);
4b complementary strand DNA of the target base sequence (4);
5 sample DNA;
6 fluorescent labels;
7 DNA chip reader;
8 probe DNA groups;
9 DNA chip;
10 hybridization solution;
11 lamps;
12 scanned images;
13 glass;
14 DNA groups;
15 c-probe DNA group;
16 c-DNA chip;
17 magnetic beads;
18 Magnet

Claims (3)

A method for detecting a target base sequence in sample DNA or RNA, comprising the following steps:
(1) Sample DNA or RNA and probe DNA or RNA are contacted in an aqueous solution to form a hybridization complex, and hybridization is performed without immobilizing either sample DNA or RNA and probe DNA or RNA. Perform;
(2) isolating the hybridization complex;
(3) dissociating the complex to separate probe DNA or RNA;
(4) The probe DNA or RNA is identified by hybridization with a DNA or RNA chip on which the complementary DNA group or RNA group of the probe DNA or RNA is immobilized, and the target base sequence in the sample DNA or RNA is determined. To detect;
Only including, a method in which a plurality of probe DNA in one sample DNA is characterized by hybridizing. The method according to claim 1, wherein plural types of probe DNA or RNA are used . The method according to claim 1 or 2 , wherein the probe DNA or RNA is fluorescently labeled.

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