JP5572939B2 - DNA sequencing method - Google Patents

Description

The present invention relates to a method for determining a DNA base sequence, and more particularly, to a method for determining a DNA base sequence using a cycle sequence reaction.

As a method for determining the base sequence of DNA, there is a base sequence determination method using a cycle sequence reaction. The cycle sequence reaction is a reaction in which a deoxy reaction and PCR are combined, and a group of DNA fragments having different chain lengths complementary to the DNA for which the base sequence is determined is obtained. This DNA fragment group is separated by electrophoresis according to the difference in chain length, and the base sequence of the target DNA is determined by, for example, irradiating laser light and detecting fluorescence having different wavelengths for each base.

More specifically, in the cycle sequence reaction, a DNA (template DNA) for the purpose of determining a base sequence is used as a primer complementary to a part of the template DNA, a heat-resistant DNA polymerase, and a reaction substrate as four kinds of bases. In addition to a suitable buffer containing every deoxyribonucleoside triphosphate (dNTPs), every four bases dideoxyribonucleoside triphosphates (ddNTPs) and metal ions, DNA denaturation step, primer annealing step to DNA, In addition, the temperature cycle step consisting of the step of extending the complementary strand of the template DNA is repeated several tens of times. Here, in the extension step, the reaction substrate is incorporated into the 3 ′ end of the complementary strand of the template DNA by the thermostable DNA polymerase and the complementary strand is extended. However, when ddNTPs are incorporated as the reaction substrate, the complementary strand is extended. Stops. As a result, a group of DNA fragments having different chain lengths is obtained as a cycle sequence reaction product.

In recent years, analysis of cycle sequence reaction products has been carried out by using capillary electrophoresis apparatuses and electrophoresis apparatuses using microchips in which capillary channels are formed in a substrate, and separation and separation using these so-called capillary electrophoresis apparatuses. Detection has become mainstream. In Patent Document 1, in a DNA base sequence determination method in which separation and detection are performed by electrophoresis using an acrylamide gel plate, in order to increase detection signal intensity, DNA polymerase modified with amino acid and fluorescein fluorescent dye are used. Each labeled ddNTPs is further disclosed to further optimize the mixed molar ratio of dNTPs and ddNTPs. On the other hand, when a capillary electrophoresis apparatus is used, since a very small amount of sample is used for electrophoresis as compared with gel plate electrophoresis, further measures for increasing the signal intensity are required.

JP 9-271400 A

Accordingly, in view of the above problems, the object of the present invention is to provide a DNA base sequence determination method using a cycle sequence reaction, particularly when a cycle sequence reaction product is analyzed using a capillary electrophoresis apparatus. It is to provide a method for determining the base sequence of DNA.

As a result of intensive studies, the inventor performs heat treatment and cooling treatment on a sample solution containing DNA whose base sequence is to be determined, and then mixes substances necessary for the cycle sequence reaction, and performs the cycle sequence reaction. Thus, the inventors have found that the object of the present invention can be achieved, and have completed the present invention.

The present invention includes the following inventions.
(1) Mixing substances and base sequences necessary for a cycle sequence reaction including a primer, deoxyribonucleoside triphosphates for each of four types of bases, dideoxyribonucleoside triphosphates for each of four types of bases, and a heat-resistant DNA polymerase In the presence of the DNA to be determined, a cycle sequence reaction is performed to amplify the DNA fragment group of the complementary strand of the DNA whose base sequence is to be determined. In the DNA sequencing method for determining the nucleotide sequence,
(I) a step of heat-treating a sample solution containing DNA whose base sequence is to be determined at a temperature of 80 ° C. or higher and lower than 100 ° C .;
(Ii) the heat-treated sample solution, 0 ° C. or higher, cooling processing step, characterized in that the cooling in the heat treatment within one minute after the end temperature of 10 ° C. or less and,
(Iii) a step of adding a mixed substance necessary for the cycle sequence reaction to the cooled sample solution to perform the cycle sequence reaction;
DNA sequencing method comprising
(2) The DNA base sequencing method according to (1), wherein the heat treatment is held at a heating temperature for 10 seconds to 10 minutes.
( 3 )
When the primer is a primer composed only of nucleotides, among the mixed substances necessary for the cycle sequencing reaction, the primer is added to the sample solution in the step (i) and subjected to heat treatment (1) or (2 The method for determining the base sequence of DNA according to any one of the above.

In the present invention, in the DNA sequencing method using cycle sequencing reaction, the DNA to be sequenced, that is, the template DNA, is heated at a temperature of 80 ° C. or higher and lower than 100 ° C. Signal intensity can be increased when analyzing reaction products. This is because the template DNA can be sufficiently dissociated and denatured by subjecting the template DNA to such a heat treatment, and the annealing efficiency of the primer in the annealing step of the cycle sequence reaction is increased. As a result, the cycle sequence is increased. This is thought to increase the yield of the reaction product. Furthermore, the heat treatment of the present invention is performed on the template DNA in the absence of a heat-resistant DNA polymerase, a substrate labeled with a fluorescent dye, or a primer labeled with a fluorescent dye. Therefore, sufficient heat treatment can be performed on the template DNA without degrading enzyme activity due to heating and without causing degradation of the fluorescent dye.

In order to increase the signal intensity in the analysis of the cycle sequence reaction product, it is conceivable to increase the amount of template DNA or increase the number of temperature cycles in the cycle sequence reaction. However, when the amount of template DNA is increased, when analyzing reaction products by electrophoresis, there are problems that the resolution decreases, noise increases, and the background increases. In addition, when the number of temperature cycles is increased, an increase in reaction time is unavoidable, and there is a risk of undesirable amplification of by-products. On the other hand, in the DNA sequencing method according to the present invention, only the heat treatment is performed on the template DNA, and the signal in the analysis of the cycle sequence reaction product is not accompanied by a decrease in resolution and an increase in noise and background. It is possible to increase the strength.

The DNA for which the base sequence to be used in the present invention should be determined may be prepared as the DNA for which sequencing is intended, incorporated into a vector suitable for base sequence determination, such as an M13 phage vector or a pUC-based plus vector. it can. Hereinafter, a vector in which DNA for sequencing is incorporated is sometimes simply referred to as template DNA.

The mixed substances necessary for the cycle sequencing reaction include at least a primer, deoxyribonucleoside triphosphates for every four bases, dideoxyribonucleoside triphosphates for every four bases, and a thermostable DNA polymerase.

As a primer, an oligonucleotide complementary to a part of the template DNA upstream of the DNA region 3 'intended for base sequence determination is used. The number of bases of the primer is, for example, about 10 to 30, and it does not matter whether it is an oligonucleotide synthesized by a well-known method or a naturally derived oligonucleotide. The primer is not limited to a primer composed only of nucleotides, and a labeled primer in which the 5 'end is labeled with various fluorescent substances can be used (Dye Primer method).

As the thermostable DNA polymerase, it is preferable to use a thermostable DNA polymerase lacking 5 ′ → 3 ′ exonuclease activity. More preferably, a thermostable DNA polymerase lacking both 5 '→ 3' exonuclease activity and 3 '→ 5' exonuclease activity is used. Specific examples include Stoffel fragment, Bca BEST (registered trademark) DNA polymerase (Takara), ΔTth (registered trademark) DNA polymerase (Toyobo), Gene Taq DNA polymerase, ΔTaq (registered trademark) DNA polymerase, and the like. It is done.

As substrates for the cycle sequencing reaction, deoxyribonucleoside triphosphates (dATP, dCTP, dGTP and dTTP; hereinafter collectively referred to as dNTPs) for each of four bases, and dideoxyribonucleosides that are analogs of each dNTP Prepare triphosphate (ddATP, ddCTP, ddGTP, and ddTTP; hereinafter collectively referred to as ddNTPs). Here, as the ddNTPs, labeled ddNTPs labeled with various fluorescent substances can be used. The fluorescent substance may be labeled with a fluorescent substance that emits fluorescence of different wavelengths for each of the four types of bases, or may be labeled with a fluorescent substance that emits fluorescence of the same wavelength (Dye Terminator method).

As a solvent for dissolving the mixed substances necessary for the cycle sequence reaction, an appropriate buffer solution that can be adjusted within the optimum pH range of the polymerase activity of the thermostable DNA polymerase used by the pH of the reaction system during the cycle sequence reaction Is used. As the buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES-KOH buffer, phosphate buffer, and the like can be used. The reaction system at the time of the cycle sequence reaction contains template ^ions , mixed substances necessary for the cycle sequence reaction, and metal ions such as Mg ²⁺ and K ⁺ . Furthermore, an SH reducing agent such as 2-mercaptoethanol or dithiothreitol may be added as appropriate in order to increase the polymerase activity.

The practitioner can appropriately adjust each concentration of the template DNA in the solution for performing the cycle sequence reaction and the concentration of the mixed substance necessary for the cycle sequence reaction.

A commercially available cycle sequence reaction reagent kit can be used as a mixed substance necessary for the cycle sequence reaction. For example, as a dye terminator sequencing kit, BigDye (registered trademark) Terminators v1.1 Cycle Sequencing Kit, BigDye Terminator v3.1 Cycle Sequencing Kit, dRhodamine Terminator Cycle Sequencing Kits, dGTP BigDye Terminator Cycle Sequencing Kits (Applied Biosystems) DYEnamic ET Terminator Cycle Sequencing Kit (manufactured by GE Healthcare). Examples of the Dye Primer method cycle sequencing kit include BigDye Primer Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and Thermo Sequenase Primer Cycle Sequencing Kit (GE Healthcare).

Next, the operation steps of the DNA base sequence determination method of the present invention will be described. FIG. 1 shows a flowchart of a DNA base sequence determination method according to an embodiment of the present invention, in which a sample solution containing DNA whose base sequence is to be determined is heated (step 1; S1), and the heat-treated sample Step of cooling the solution (Step 2; S2), Step of adding a mixed substance necessary for the cycle sequence reaction to the cooled sample solution (Step 3; S3), and performing the cycle sequence reaction (Step 4; S4) And a step of analyzing the cycle sequence reaction product (step 5; S5).

[S1. Heat treatment process]
The sample solution containing the DNA whose base sequence is to be determined is subjected to a heat treatment by maintaining it at a temperature of 80 ° C. or higher and lower than 100 ° C. Here, less than 100 ° C. means that the sample solution may be in a state where it does not boil, and is preferably 99 ° C. or less. As a solvent for the sample solution, pure water or a suitable buffer, for example, the solvent for the cycle sequence reaction described above can be used. The heat treatment operation may be performed, for example, by placing a reaction tube containing the sample solution in a warm water bath, or by temperature control using a thermal cycler. As a condition for the heat treatment, the heating temperature is more preferably maintained for 10 seconds or more and 10 minutes or less. More preferably, the temperature is maintained at 95 ° C. or higher and lower than 100 ° C. for 10 seconds or longer and within 5 minutes. The heat treatment can be sufficiently denatured with respect to the template DNA having a complicated three-dimensional structure by performing the heating at 80 ° C. or more, and is particularly effective when a double-stranded circular template DNA is prepared. Since the melting temperature (Tm) of general DNA is in the range of 70 ° C., it is considered effective to make DNA denatured by heating at 80 ° C. or higher. By setting the heating temperature to less than 100 ° C., DNA damage due to heat can be prevented. In particular, when the heat treatment temperature is 95 ° C. or higher, the template DNA can be dissociated and denatured in a short time.

Here, in this heat treatment step, a mixed substance necessary for the cycle sequence reaction is not included in the sample solution. However, when using a primer consisting only of an oligonucleotide that is not a labeled primer, it may be added to the sample solution. Therefore, the template DNA can be sufficiently denatured while preventing a decrease in the activity of the heat-resistant DNA polymerase and the decomposition of the labeled substance such as a fluorescent dye bound to the labeled primer or the labeled ddNTPs. It should be understood that substances that are not affected by decomposition, such as metal ions and SH reducing agents, may be included in the sample solution in this heat treatment step.

[S2. Cooling process]
The sample solution after the heat treatment is cooled. As a condition of the cooling treatment, it is preferable to cool rapidly after the heat treatment, and the cooling temperature is 0 ° C. or higher and 10 ° C. or lower, more preferably 0 ° C. or higher and 5 ° C. or lower, within 1 minute after the heat treatment is completed. It is preferable to cool. It is considered that the template DNA can be kept in a denatured state by rapidly cooling the sample solution to 10 ° C. or lower after the heat treatment. As the operation of the cooling step, the reaction tube containing the sample solution may be placed in an ice water bath or an ice bath, or the cooling treatment may be performed by temperature control using a thermal cycler. For sample solutions on the order of tens of microliters, put the reaction tube into an ice-water bath immediately after the heat treatment, or set the temperature of the thermal cycler to the cooling temperature to reach the cooling temperature within a few seconds to one minute. The sample solution can be kept. When a thermal cycler is used, the above-described heat treatment step, cooling treatment step, and subsequent cycle sequence reaction step can be performed consistently by the thermal cycler, which is a simple operation.

[S3 and S4. A process of adding a mixture substance necessary for the cycle sequence reaction and performing the cycle sequence reaction]
To the sample solution subjected to the cooling treatment, mixed substances necessary for the cycle sequence reaction, that is, primers, heat-resistant DNA polymerase, dNTPs, and ddNTPs are added. When the cycle sequence reaction solvent is not used in the sample solution prepared in the preceding heat treatment step, a circle sequence reaction solvent is added to adjust the pH to be suitable for the cycle sequence reaction. Regarding the primer, as described above, when the above-described labeled primer is not used, that is, when a primer consisting of only nucleotides is used, it may be added to the sample solution in the heat treatment step and subjected to heat treatment together with the target DNA. Good. Substances that are not affected by decomposition or the like due to heating, such as metal ions and SH reducing agents, are added during the cycle sequence reaction step if they have not been added to the sample solution subjected to the heat treatment step. As the addition operation, the sample solution subjected to the cooling treatment, the mixed substance necessary for the cycle sequence reaction, and other substances to be added may be mixed, and the order of addition between the solutions is not limited.

As a temperature condition of the cycle sequence reaction, a temperature cycle consisting of a denaturation step, an annealing step and an extension step is usually 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 2 minutes. In the first denaturation step (preheating), it is possible to set a longer time of about 1 to 10 minutes in order to sufficiently denature the template DNA. Through this cycle sequence reaction step, a group of DNA fragments having a different chain length that is complementary to the DNA whose base sequence is to be determined is synthesized as a cycle sequence reaction product.

Thus, in the present invention, since the cycle sequence reaction is performed on the heat-treated and cooled template DNA, the template DNA is sufficiently denatured, and the primer easily approaches the template DNA in the annealing step. Therefore, it can be considered that the annealing efficiency is improved. As a result of the improvement of the cycle sequence reaction efficiency, it is considered that the yield of the cycle sequence reaction product is improved.

After the cycle sequence reaction step, the cycle sequence reaction product is purified. As a purification method, an operator can select and use suitably, such as an ethanol precipitation method and gel filtration. Moreover, a commercially available DNA purification kit can be used.

[S5. Analysis of cycle sequence reaction products]
Analysis of the obtained cycle sequence reaction product was performed by separating DNA fragment groups with different chain lengths by electrophoresis, labeling from the labeled primer in the dye primer method, and labeling from the labeled ddNTPs in the dye terminator method. A fluorescent signal from the substance is detected, and a base sequence is determined based on the detection signal. The electrophoresis method is not particularly limited, and a capillary electrophoresis apparatus can be used in addition to electrophoresis using a denaturing polyacrylamide gel capable of separating a difference of one base. Specifically, for example, RISA (registered trademark, manufactured by Shimadzu Corporation) which is a capillary electrophoresis apparatus, and DeNOVA (registered trademark) -5000HT (manufactured by Shimadzu Corporation) can be used as a microchip electrophoresis apparatus.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

As DNA for sequencing purposes, 300 ng (50 ng / μl × 6 μl) of pUC18 DNA, 3.2 pmol (1 pmol / μl × 3.2 μl) of primer (5′-CGCCAGGGTTTCCCAGTCACGAC-3 ′: SEQ ID NO: 1), BigDye ( The sample solution was prepared in a reaction tube by mixing with 3.5 μl of Sequencing Buffer attached to Cycle Sequencing Kit (registered trademark) Terminator v3.1 and 6.3 μl of MilliQ water. The sample solution was heat treated by placing the reaction tube in a 98 ° C. warm bath for 3 minutes, and then immediately cooled by placing the reaction tube in an ice water bath. By this cooling treatment, it is considered that the sample solution was cooled to the ice water temperature within 1 minute from the heating temperature. 1 μl of BigDye Terminator v3.1 Cycle Sequencing Kit Ready Reaction Premix was added to the sample solution subjected to this cooling treatment to prepare a mixed solution for cycle sequence reaction in a total amount of 20 μl. For this mixture, after a preheating step at 96 ° C. for 1 minute, at 96 ° C. for 10 seconds (denaturation step), at 50 ° C. for 5 seconds (annealing step), and at 60 ° C. for 2 minutes (extension step) The cycle sequence reaction was carried out by repeating the following temperature cycle 40 times.

The cycle sequence reaction product was purified using a DNA purification kit AutoSeq G-50 (GE Healthcare Bioscience), and the purified and recovered solution was diluted with a sample diluent P3E loading buffer (manufactured by Shimadzu Corporation) and subjected to electrophoresis. . For electrophoresis, a microchip electrophoresis apparatus was used. The electrophoresis conditions are as follows.
Microchip: Quartz plate separation medium with a 90μm width x 40μm depth channel inside: Modified P3E electrophoresis polymer (manufactured by Shimadzu Corporation)
Running buffer: P3E running buffer (manufactured by Shimadzu Corporation)
Sample injection conditions: 50V / cm, 10sec
Electrophoretic conditions: 225V / cm, 60 ° C

[Comparative example]
As a comparative example, in Example 1, the cycle sequence reaction and the electrophoresis were performed under the same conditions as in the example except that the heat treatment and the cooling treatment were not performed.

FIG. 2 a) shows the electropherogram obtained in the comparative example, and b) shows the electropherogram obtained in the example of the present invention. The horizontal axis is the number of scans, and the vertical axis is the signal intensity. As is apparent from the figure, the signal strength is low in a), whereas b) in this embodiment has a high signal strength and a high SN ratio. As described above, it was confirmed that the DNA base sequence determination method in the present invention can dramatically increase the signal intensity even in the case of the preheating usually performed in the cycle sequence reaction.

It is a flowchart which shows the base sequence determination method of DNA which is one Embodiment of this invention. a) is the electropherogram obtained in the comparative example, and b) is the electropherogram obtained in the example of the present invention.

Claims (3)

Determining the mixture and base sequence required for the cycle sequence reaction including primer, deoxyribonucleoside triphosphate per 4 bases, dideoxyribonucleoside triphosphate per 4 bases, and thermostable DNA polymerase In the presence of DNA, perform a cycle sequence reaction that synthesizes a DNA fragment group of complementary strands of the DNA whose base sequence is to be determined, and determine the base sequence by analyzing the DNA fragment group synthesized by the cycle sequence reaction. In the nucleotide sequence determination method of
(I) a step of heat-treating a sample solution containing DNA whose base sequence is to be determined at a temperature of 80 ° C. or higher and lower than 100 ° C .;
(Ii) the heat-treated sample solution, 0 ° C. or higher, cooling processing step, characterized in that the cooling in the heat treatment within one minute after the end temperature of 10 ° C. or less and,
(Iii) a step of adding a mixed substance necessary for the cycle sequence reaction to the cooled sample solution to perform the cycle sequence reaction;
DNA sequencing method comprising The DNA base sequence determination method according to claim 1, wherein the heat treatment is held at a heating temperature for 10 seconds to 10 minutes. The primer according to claim 1 or 2 , wherein when the primer is a primer composed only of nucleotides, the primer is added to the sample solution in the step (i) among the mixed substances necessary for the cycle sequencing reaction, and is subjected to heat treatment. A method for determining the base sequence of DNA according to any one of the above.

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