Nucleotide sequence identification method

Abstract

Provided is a nucleotide sequence identification method for analyzing double-stranded DNA in a single reaction system while keeping down costs and labor. A method for identifying a nucleotide sequence in a complementary pair of target polynucleotides comprising a double strand, the method having a step for identifying the nucleotide sequence in the one target polynucleotide using a first primer that includes a target recognition site that forms a complementary pair with part of one target polynucleotide of the target polynucleotide complementary pair and a step for identifying the nucleotide sequence in the other target polynucleotide using a second primer that includes a target recognition site that forms a complementary pair with part of the other target polynucleotide of the target polynucleotide complementary pair and a reaction stopping site for stopping a nucleotide elongation reaction, and the second reaction product obtained by elongation of the second primer having lower mobility than the first reaction product obtained by elongation of the first primer.

Description

[Technical field]

[0001] The present invention relates to a method for identifying a nucleotide sequence. [Background technology]

[0002] Among polynucleotides, a molecule in which deoxyribose is linked in a chain shape is called a DNA strand. Generally, in living organisms, one DNA strand is helically entangled with another DNA strand composed of complementary bases to form a double-stranded structure. The complementary bases are adenine (A) and thymine (T), and cytosine (C) and guanine (G). Each complementary base is paired through hydrogen bonds. For convenience, one strand is called the top strand and the other the bottom strand in this specification. The procedure of obtaining base sequence information of a polynucleotide by analysis is called sequencing.

[0003] Polynucleotide sequence information is used to identify pathogens, detect cancer-related mutations in genomic DNA, and predict drug resistance, efficacy, and prognosis. Given the impact on the subject's health, the information obtained by analysis must be highly accurate.

[0004] As mentioned above, once one base pair is determined, the other can be uniquely identified. Therefore, in principle, sequencing only needs to be performed on one strand. However, in practice, accuracy can decrease due to various factors such as equipment malfunctions and handling errors. Therefore, in order to improve the accuracy of sequence information, each strand of double-stranded DNA is sometimes sequenced.

[0005] For example, Patent Document 1 discloses a method for determining a consensus sequence of nucleotides in a DNA segment to be analyzed, the method including the steps of covalently linking the ends of the top and bottom strands of the DNA segment to be analyzed and sequencing both the top and bottom strands using a polymerase-based sequencing method.

[0006] Furthermore, Patent Document 2 discloses a method for determining the sequence of one or more nucleotides in a target polynucleotide, in which a target-specific primer comprises a target-binding segment and a mobility-reducing moiety that does not bind to the target.

[0007] Furthermore, Patent Document 3 discloses that tails consisting of polynucleotides of different lengths are used to reduce the mobility of PCR products, thereby enabling multiple gene loci to be analyzed at the same time. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2011-515102 [Patent Document 2] Special Publication No. 2002-536980 [Patent Document 3] U.S. Patent No. 6,197,510 Summary of the Invention [Problem to be solved by the invention]

[0009] The technology disclosed in Patent Document 1 creates a molecule in which one end of a double-stranded DNA is connected by a hairpin loop, or a circular molecule in which both ends are connected by hairpin loops. However, creating such a molecule requires cumbersome work such as linking the molecule via the hairpin loop and purifying it. The technology described in Patent Document 2 and Patent Document 3 is a method for analyzing one strand of a double-stranded DNA.

[0010] An object of the present invention is to provide a nucleotide sequence determination method for analyzing double-stranded DNA in a single reaction system while reducing costs and labor. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the present invention provides a nucleotide sequence identification method for identifying one or more nucleotide sequences in a double-stranded target polynucleotide complementary pair, the method comprising the steps of: identifying one or more nucleotide sequences in one target polynucleotide using a first primer including a target recognition site that forms a complementary pair by hydrogen bonding with a portion of one target polynucleotide of the target polynucleotide complementary pair; and identifying one or more nucleotide sequences in the other target polynucleotide using a second primer that includes a target recognition site that forms a complementary pair by hydrogen bonding with a portion of the other target polynucleotide of the target polynucleotide complementary pair, and a reaction termination site that terminates a nucleotide extension reaction by DNA polymerase, wherein a second reaction product obtained by extending a complementary strand on the other target polynucleotide with the second primer has a smaller mobility than a first reaction product obtained by extending a complementary strand on the one target polynucleotide with the first primer. Effect of the Invention

[0012] According to the present invention, a nucleotide sequence determination method for analyzing double-stranded DNA in a single reaction system while reducing costs and labor can be provided. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram showing the concept of a nucleotide sequence determination method according to embodiment 1. [Diagram 2] 1 is a flow chart showing the procedure for identifying a nucleotide sequence. [Diagram 3] FIG. 1 shows primer information. [Figure 4] 11 is a flowchart showing a procedure for confirming effects. [Diagram 5] FIG. 1 shows temperature conditions for cycle sequence reaction. [Figure 6] Schematic diagram of DNA and primers used in the Examples. [Figure 7] Electropherograms obtained as a result of electrophoresis for the Examples and Comparative Examples. [Figure 8] FIG. 13 is a conceptual diagram of DNA and primers in embodiment 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In this specification, the DNA strand whose sequence is to be determined is called the analysis target. The analysis target can be prepared by incorporating it into an M13 phage vector or a plasmid. Alternatively, a part of a DNA fragment created by PCR may be used as the analysis target. The entire DNA strand including the analysis target is called the target polynucleotide. Below, a method for identifying one or more nucleotide sequences in a target polynucleotide complementary pair that constitutes a double strand will be described with reference to four embodiments. Embodiment 1

[0015] The first embodiment will be described with reference to Figures 1 to 7. Figure 1 is a diagram showing the concept of the method for identifying a nucleotide sequence according to the first embodiment.

[0016] Cycle sequencing reactions require at least the following reagents: a target polynucleotide, deoxyribonucleoside triphosphates for each of the four bases (adenine (A), guanine (G), cytosine (C), and thymine (T)), dideoxyribonucleoside triphosphates for each of the four bases, a thermostable DNA polymerase, and two types of primers, which are described below.

[0017] Here, two types of primers used in this embodiment will be described. For convenience, one primer is called the first primer 203, and the other primer is called the second primer 204. The first primer 203 and the second primer 204 contain bases complementary to a part of the target polynucleotide 202. As shown in FIG. 1, the first primer 203 and the second primer 204 are designed so that the 3' side of the DNA faces the analysis target 201. Therefore, the first primer 203 and the second primer 204 form a complementary pair with different DNA strands. The complementary bases are usually continuous for about 10-30 bases. Then, the first primer 203 is used to identify the nucleotide sequence in one target polynucleotide, and the second primer 204 is used to identify the nucleotide sequence in the other target polynucleotide.

[0018] Furthermore, in this embodiment, the second reaction product obtained by the second primer 204 extending a complementary strand on the other target polynucleotide is made to have a smaller mobility than the first reaction product obtained by the first primer 203 extending a complementary strand on one target polynucleotide. As a result, when electrophoresed, the second reaction product is always detected later than the first reaction product, so that double strands can be analyzed in one reaction system, making it possible to improve the efficiency of the analysis. The specific configuration of each primer will be described below with reference to FIG. 1.

[0019] First, the first primer 203 has a target recognition site 2030 that forms a complementary pair by hydrogen bonding with a portion of one target polynucleotide of a target polynucleotide complementary pair.

[0020] In contrast, the second primer 204 has a target recognition site 2040 that forms a complementary pair by hydrogen bonding with a portion of the other target polynucleotide of the target polynucleotide complementary pair, a reaction termination site 2041, and a mobility reducing site 2042.

[0021] The reaction stop site 2041 is located on the 5' side of the target recognition site 2040 of the second primer 204, and contains a compound that stops the extension reaction of a nucleotide by a thermostable DNA polymerase. Typical examples of compounds that constitute the reaction stop site 2041 include inosine, ribonucleosides, amino acid residues, polyethylene glycol, etc. The reaction stop site 2041 is linked to a mobility reducing site 2042.

[0022] The mobility reducing portion 2042 is located on the 5' side of the reaction termination portion 2041 of the second primer 204, and serves to reduce the migration speed of the reaction product. For convenience, the DNA fragment group extended from the 3' end of the first primer 203 is called the first fragment group, and the DNA fragment group extended from the 3' end of the second primer 204 is called the second fragment group. That is, the mobility reducing portion 2042 is composed of a compound that reduces the migration speed of the second fragment group in the separation medium during electrophoresis. More specifically, the mobility reducing portion 2042 slows down the migration speed of the molecule with the smallest chain length in the second fragment group compared to the migration speed of the molecule with the largest chain length in the first fragment group. The mobility reducing portion 2042 can be made of a polynucleotide, an amino acid residue, polyethylene glycol, or the like.

[0023] Next, the length of the second primer 204 will be described taking as an example a case where a polynucleotide is used as the mobility reducing portion 2042. First, since the second primer 204 is longer than the first primer 203, the mobility of the reaction product derived from the second primer 204 is smaller than the mobility of the reaction product derived from the first primer 203, making it easier to distinguish and detect each reaction product. The mobility of the reaction product derived from the second primer 204 can be adjusted with high precision by appropriately setting the length of the mobility reducing portion 2042.

[0024] In addition, when the region sandwiched between the two primers, specifically, the chain length from the 5' end of the target recognition site of the first primer 203 to the 5' end of the target recognition site of the second primer 204, is defined as the amplification region 205 (see FIG. 1), it is desirable to make the mobility reduction region 2042 longer than the amplification region 205. If the mobility reduction region 2042 does not have a chain length equal to or longer than the amplification region 205, peaks will overlap in a part of the region of the first fragment group and the second fragment group, making it difficult to distinguish the peaks in the region. On the other hand, when the mobility reduction region 2042 is a polynucleotide having a chain length equal to or longer than the amplification region 205, the analysis accuracy is improved. Furthermore, when the mobility reduction region 2042 is longer than the amplification region, a gap will be generated between the first fragment group and the second fragment group, which has the advantage of making it easier to distinguish the boundary between the fragment groups.

[0025] Furthermore, it is desirable that the polynucleotide constituting the mobility-reducing portion 2042 does not have a region in which bases complementary to the target polynucleotide are consecutive. If such a region exists, the mobility-reducing portion 2042 will start annealing in that region, which will become a cause of noise. In order to eliminate such a region, it is effective to make the thermal stability of the mobility-reducing portion 2042 when it forms a complementary pair smaller than the thermal stability of the target recognition portion 2040 when it forms a complementary pair.

[0026] Next, a method for identifying a nucleotide sequence using the above-mentioned primers will be specifically described. Figure 2 is a flow chart showing the procedure for identifying a nucleotide sequence.

[0027] As shown in FIG. 2, first, a cycle sequence reaction shown in step S101 is carried out.

[0028] Here, the reaction system of the cycle sequence reaction will be described. In the cycle sequence reaction, in addition to the first primer 203, the second primer 204, the target polynucleotide, and the heat-resistant DNA polymerase, deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP; hereinafter collectively referred to as dNTPs) for each of the four bases, and dideoxyribonucleoside triphosphates (ddATP, ddCTP, ddGTP, and ddTTP; hereinafter collectively referred to as ddNTPs) which are analogs of each dNTP are prepared as substrates. Here, the ddNTPs may be labeled ddNTPs labeled with various fluorescent substances. The fluorescent substances are labeled with fluorescent substances which emit fluorescence of different wavelengths for each of the four bases. As a solvent for dissolving the mixed substances required for the cycle sequence reaction, a buffer solution is used which can adjust the pH of the reaction system during the cycle sequence reaction to within the optimal pH range for the polymerase activity of the heat-resistant DNA polymerase used. As the buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES-KOH buffer, phosphate buffer, etc. can be used. The reaction system of the cycle sequencing reaction contains the target polynucleotide, the mixture necessary for the cycle sequencing reaction, and metal ions such as Mg2+ and K+. Furthermore, SH reducing agents such as 2-mercaptoethanol and dithiothreitol may be added as appropriate to enhance the polymerase activity. The operator can adjust the respective concentrations of the target polynucleotide and the mixture necessary for the cycle sequencing reaction in the solution in which the cycle sequencing reaction is performed as appropriate. As the mixture necessary for the cycle sequencing reaction, a commercially available cycle sequencing reaction reagent kit can be used. For example, BigDye(TM) Terminators v1.1 Cycle Sequencing Kit, BigDye(TM) Terminator v3.1 Cycle Sequencing Kit, dRhodamine Terminator Cycle Sequencing Kits, dGTP BigDye(TM) Terminator Cycle Sequencing Kits, etc., from Applied Biosystems(TM) can be mentioned.

[0029] Next, each step in the cycle sequencing reaction will be described. The compounds necessary for the cycle sequencing reaction described above are mixed. As for the primers, as described above, two types of primers are used. In the cycle sequencing, a temperature cycle consisting of a step of converting double-stranded DNA into a single strand (hereinafter referred to as a denaturation step), a step of forming a complementary pair by hydrogen bonding a part of a complementary region of a target polynucleotide with a primer (hereinafter referred to as an annealing step), and a step of extending the complementary strand by adding dNTP or ddNTP to the 3' end of each primer using a heat-resistant DNA polymerase (hereinafter referred to as an extension step) is repeated about 25 to 40 times. In general, the denaturation step is performed at 96°C for 10 seconds, the annealing step is performed at 50°C for 5 seconds, and the extension step is performed at 60°C for 4 minutes. In the first denaturation step (preheating), a relatively long time of about 1 to 10 minutes can be set in order to sufficiently denature the template DNA.

[0030] This cycle sequencing reaction synthesizes a group of DNA fragments of different lengths that are complementary to the DNA to be sequenced as reaction products. The chain lengths of the first group of fragments derived from the first primer 203 are always shorter than the chain lengths of the second group of fragments derived from the second primer 204.

[0031] After the cycle sequencing reaction, a purification process (step S102) is performed. The purpose of the purification process is to exchange the solvent for one suitable for electrophoresis and to remove unreacted dNTPs, ddNTPs, and primers. The purification method may be selected appropriately by the operator, such as ethanol precipitation or gel filtration. A commercially available DNA purification kit may also be used.

[0032] The purified reaction products are separated and detected by electrophoresis (step S103). DNA fragments are separated by the molecular sieve effect of the separation medium, and fluorescent signals from the labeled substances derived from the labeled ddNTPs are detected. Then, the base sequence is determined (step S104) based on the detection signals. The type of electrophoresis method is not particularly limited. In addition to electrophoresis using a denaturing polyacrylamide gel that can separate differences of one base, a capillary electrophoresis device can be used. The following describes the case where a capillary electrophoresis device is used as an example.

[0033] In this embodiment, the migration speed of the first fragment group is always greater than that of the second fragment group. Therefore, as shown in FIG. 1, a signal 206 derived from the first fragment group is always detected earlier than a signal 207 derived from the second fragment group. As a result, sequence information of a double-stranded DNA can be obtained by one cycle sequencing reaction and electrophoresis. In addition, in this embodiment, fluorescent substances with different wavelengths are used for each of the four types of bases, so that fluorescent signals can be detected with one capillary, leading to improved workability and processing capacity.

[0034] Next, the effect of the embodiment was confirmed using an actual sample. Figure 3 shows primer information, Figure 4 is a flow chart showing the procedure for confirming the effect, and Figure 5 shows the temperature conditions of the cycle sequence reaction. The following will be described in two parts: an example and a comparative example.

[0035] [Example] First, an example will be described. Figure 6 is a conceptual diagram of DNA and primers used in the example.

[0036] As the target polynucleotide, 5 ng of pUC18 DNA was amplified with primer F1 (corresponding to the first primer 203) and primer R1 shown in FIG. 3 to obtain a PCR fragment 502 (step S401). Next, the PCR fragment was purified with a DNA purification column (step S402) and diluted with TE buffer to 1 ng / μl. Then, 1 μl (1 ng) of the PCR fragment was used as the target polynucleotide, and 1 μl (4 pmol) each of primer F1 and primer R2 (corresponding to the second primer 204), 2 μl of Sequencing Buffer included with BigDye(TM) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems(TM)), 4 μl of BigDye(TM) Terminator 3.1 Ready Reaction Mix, and 11 μl of MilliQ water were added and mixed in a 0.2 ml reaction tube. At this time, as shown in Fig. 6, the 3' side of each of the DNA of primer F1 and primer R2 faces the analysis target 501. Furthermore, the reaction tube was loaded into a thermal cycler, and a cycle sequencing reaction consisting of a denaturation step, an annealing step, and an extension step was carried out under the conditions shown in Fig. 5 (step S403).

[0037] Next, the reaction product was purified by ethanol precipitation (step S404). In the ethanol precipitation, 2 μl of 125 mM EDTA-2Na (pH 8.0), 2 μl of 3 M sodium acetate (pH 5.0), 1 μl (100 ng) of pUC18 DNA for coprecipitation, and 50 μl of 99.5% ethanol were added to the reaction tube and stirred. Since the pUC18 DNA for coprecipitation is not fluorescently labeled, it is not detected during electrophoresis. The reaction tube was left to stand at room temperature for 15 minutes to aggregate the DNA, and then centrifuged at 4°C and 2000g for 45 minutes. After centrifugation, the supernatant was discarded, 70 μl of 70% ethanol was added, and the mixture was centrifuged at 4°C and 2000g for 15 minutes. After centrifugation, the supernatant was discarded, and the precipitated pellet-like DNA was air-dried. The DNA pellet was dissolved in 10 μl of high-purity formamide. The DNA solution was subjected to capillary electrophoresis (step S405), and the nucleotide sequence was determined (step S406).

[0038] [Comparative Example] Comparative Examples will be described. In the Comparative Examples, the primers added during the cycle sequence reaction in step S403 are different from those in the Examples. Specifically, in Comparative Example 1, a sample was prepared to which only primer F1 was added, in Comparative Example 2, a sample was prepared to which only primer R1 was added, and in Comparative Example 3, a sample was prepared to which primers F1 and R1 were added. It should be noted that primer R1 does not have a reaction termination site or a mobility reduction site. In these Comparative Examples, the cycle sequence reaction, purification, and electrophoresis were performed under the same conditions as in the Examples, except for the primers.

[0039] FIG. 7 shows electropherograms obtained as a result of electrophoresis for the Examples and Comparative Examples, with the horizontal axis representing the number of scans (time) and the vertical axis representing signal intensity. As is clear from FIG. 7, sequence information was obtained only for the bottom strand in Comparative Example 1, and only for the top strand in Comparative Example 2. Furthermore, although peaks for both the top strand and the bottom strand were detected in Comparative Example 3, they overlap and cannot be distinguished. In contrast, in the Examples, the peaks and sequence information for both the top strand and the bottom strand are developed on the horizontal axis, allowing for clear distinction. Embodiment 2

[0040] In the second embodiment, ddNTPs labeled with four different fluorescent substances for each of the four bases are used, but in the second embodiment, the primers are fluorescently labeled. In this embodiment, only one type of fluorescent label is required, so the cost required for fluorescent labeling can be reduced. In addition, according to this embodiment, since it is sufficient to read light of one wavelength, there is an advantage that a reading device with low performance can be used. Note that the wavelengths of the fluorescent dyes that label the first primer 203 and the second primer 204 may be the same or different.

[0041] In the second embodiment, the target polynucleotide is dispensed into four reaction tubes during the cycle sequencing reaction. Here, each tube is called the first tube, the second tube, the third tube, and the fourth tube. dNTP, the first primer 203, the second primer 204, a thermostable DNA polymerase, and a buffer solution are added to each tube. Next, ddATP is added to the first tube, ddCTP to the second tube, ddGTP to the third tube, and ddTTP to the fourth tube. After that, the steps from temperature cycle to purification are the same as those of the first embodiment.

[0042] The purified reaction products are separated and detected by electrophoresis. At this time, the samples in each tube are subjected to electrophoresis in separate flow paths. The molecular sieving effect of the separation medium separates the DNA fragments, and the fluorescent signals from each primer are detected. The base sequence is determined based on the detection signals.

[0043] In this embodiment, the migration speed of the first fragment group is always faster than that of the second fragment group. Therefore, the signal derived from the first fragment group is always detected earlier than the signal derived from the second fragment group, and they can be distinguished from each other. Furthermore, in this embodiment, the Dye Primer method, which is a modification of the Sanger method, can be used. Embodiment 3

[0044] In the second embodiment, the primer is labeled with a fluorescent dye, but in the third embodiment, the primer is labeled with a radioisotope. For example, a phosphorus isotope can be used to label the primer. 32 However, other radioisotopes may be used as long as they are detectable. In this embodiment, an electrophoresis device that does not have a laser or a fluorescence detection unit can be used.

[0045] In the first embodiment, a linear molecule is used as the mobility-reducing portion of the second primer, but in the fourth embodiment, the mobility-reducing portion of the second primer has a branched structure. FIG. 8 is a conceptual diagram of DNA and primers in the fourth embodiment. As shown in FIG. 8, a compound having a branched structure is bound to the 5' end of the second primer 204', and a part of the molecule of the second primer 204' is branched. Examples of compounds having a branched structure include oligodendrimer, branched polyethylene glycol, and the use of Poly(ADP)adenyl group.

[0046] According to this embodiment, the mobility is reduced in the branched structure, and the effect of reducing the mobility is greater than that of a molecule of the same length with a linear structure. Therefore, if the degree of the mobility reduction effect due to the branched structure can be predicted, the length of the second primer can be shortened accordingly, leading to cost reduction. In addition, even if the chain length of the amplified region is long, the mobility reduction portion can be prevented from becoming excessively long.

[0047] The present invention is not limited to the above-described embodiment, but includes various modified examples. For example, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration. [Explanation of symbols]

[0048] 201, 501: analysis target, 202: target polynucleotide, 203: first primer, 204, 204': second primer, 205: amplified region, 206: signal derived from first fragment group, 207: signal derived from second fragment group, 502: PCR fragment, 2030, 2040: target recognition site, 2041: reaction stop site, 2042: mobility reduction site

Claims

1. A method for identifying one or more nucleotide sequences in a double-stranded target polynucleotide complementary pair, comprising: using a first primer including a target recognition site that forms a complementary pair by hydrogen bonding with a portion of one of the target polynucleotides of the target polynucleotide complementary pair, identifying one or more nucleotide sequences in said one target polynucleotide; using a second primer including a target recognition site that forms a complementary pair by hydrogen bonding with a portion of the other target polynucleotide of the target polynucleotide complementary pair, and a reaction termination site that terminates a nucleotide extension reaction by a DNA polymerase; and identifying one or more nucleotide sequences in the other target polynucleotide, the reaction termination site comprises any one of inosine, a ribonucleoside, an amino acid residue, and polyethylene glycol; A method for identifying a nucleotide sequence, characterized in that, when electrophoresed, a second reaction product obtained by the second primer extending a complementary strand on the other target polynucleotide is detected later than a first reaction product obtained by the first primer extending a complementary strand on one of the target polynucleotides.

2. The method for identifying a nucleotide sequence according to claim 1, the first reaction product and the second reaction product are separated by electrophoresis and detected by fluorescence; A method for determining a nucleotide sequence, wherein the ddNTP contained in the extension reaction is labeled with a fluorescent substance.

3. The method for identifying a nucleotide sequence according to claim 1, the first reaction product and the second reaction product are separated by electrophoresis and detected by fluorescence; A method for identifying a nucleotide sequence, wherein the first primer and the second primer are labeled with a fluorescent substance.

4. The method for identifying a nucleotide sequence according to claim 1, the first reaction product and the second reaction product are separated and detected by electrophoresis; A method for identifying a nucleotide sequence, wherein the first primer and the second primer are labeled with a radioisotope.

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