KR20060030835A - DNA base sequence analysis using DNA synthesis reaction - Google Patents

Abstract

The invention consumes specific dNTPs while synthesizing DNA to identify nucleotide sequences, and proceeds with a separate DNA synthesis reaction that detects the consumption of specific dNTPs, while controlling the supply of specific dNTPs as a result of this exhaustion detection reaction. It is a way to find out the DNA sequence.

The present invention relates to a method for analyzing a DNA base sequence and to find a DNA sequence without electrophoresis. When synthesizing DNA to find the sequence in the DNA synthesis reaction for dNTP consumption, a certain amount of dNTP is supplied, and The remaining dNTP after the reaction is fed to the dNTP depletion detection DNA synthesis reaction at another point. The DNA synthesis reaction for detecting specific dNTP depletion can generate a sequence that the restriction enzyme can recognize if the specific dNTP is present. When the restriction enzyme is reacted and washed after the DNA synthesis reaction, the fluorescent dye attached to the DNA end is also washed away. Otherwise, the restriction enzyme cannot make a recognizable sequence, so it is not washed out after the restriction enzyme reaction. Therefore, a specific dye dNTP is sent to the detection DNA synthesis reaction after consumption DNA synthesis reaction to find out how much was consumed by the fluorescent dye detector. Base sequence of DNA synthesized in synthesis reaction can be determined.

For this, dGTP, dATP, dTTP, and dCTP are supplied to the DNA synthesis reaction for consumption in order, and the degree of consumption is confirmed in the DNA synthesis reaction for consumption detection. By measuring fluorescence in real time, the specific dNTP to be supplied is determined, and each dNTP is supplied in excess of 10% by the amount required to calculate the fluorescence and the degree of sequencing so that the sequence of the same base can be identified.

The most excellent method of living out to date is the use of electrophoresis, so it is difficult to identify the nucleotide sequence more than 800 fundamentally, but the present invention can be identified up to 10,000 or more according to the improvement of enzymes. Speed and accuracy can be increased by performing the same reactions simultaneously or by identifying complementary DNA strands.

DNA base sequence, electrophoresis, living method, fluorescent dye, restriction enzyme, DNA synthesis reaction for dNTP consumption, DNA synthesis reaction for dNTP consumption detection

Description

DNA sequencing by DNA synthesis using DNA synthesis

1 shows DNA synthesis reaction for dNTP consumption and DNA synthesis reaction for dNTP consumption detection.

2 shows the droplets and (-) electrode trajectory between the positive and negative plates.

3 is a droplet transported by electrowetting

4 is a reaction material supply port and reaction place and outlet

FIG. 5 shows the trajectory of the negative electrode connecting each element of FIG.

Figure 6 is a reaction material reservoir, supply port and washing water discharge needle

7 is a position of the reaction material supply port and the washing water discharge needle

8 is an electric capillary supplying a reaction material

9 is a needle for draining the wash water

10 is a detection method using a restriction enzyme and a detection method using a fluorescent material

11 is a detection method using a dNTP with a fluorescent material and a detection method using an amine reaction

12 is a detection method using hybridization of DNA with fluorescent material and a detection method using ferrocene

Fig. 13 shows the detection method using luciferin and the detection method using DNA immobilized elsewhere.                 

14 is a detection method using hybridization of DNA with enzyme and a detection method using restriction enzyme

15 is a detection method using a fluorescent antibody and a detection method using a magnetic bead

16 shows the addition of the trajectory and the wash outlet to wash the negative electrode trajectory.

17 shows the different positions of the wash buffer reservoir and the supply port.

18 shows the dispersion point of the (-) electrode trajectory

<Description of the code | symbol about the principal part of drawing>

Ports for connecting and controlling electrodes, omission of air inlet to prevent negative pressure from supply and discharge module

1: Plate with transparent positive electrode for horizontal transportation

2: Transparent (+) electrode for horizontal transport

3: hydrophobic insulator

4: Plate with negative electrode for horizontal transportation

5: fluid droplet

6: DNA strands to continue in DNA synthesis

7: biotinylated template DNA strand at 5 'end

8: biotin

9: streptavidin

10: DNA hydrophilic spot for dNTP consumption

11: (-) electrode for horizontal transportation                 

12: DNA hydrophilic spot for dGTP depletion detection

13: washing water outlet

14: wash buffer inlet

15: dGTP inlet

16: dATP inlet

17: dTTP inlet

18: dCTP inlet

19: DNA synthase injection hole

20: restriction enzyme inlet

21: DNA synthesis reaction site for dATP depletion detection

22: DNA synthesis reaction site for dTTP depletion detection

23: DNA synthesis reaction site for dCTP depletion detection

24: Washing buffer injection electrocapillary

25: reservoir

26: dGTP injection electrocapillary

27: dATP injection electrocapillary

28: dTTP injection electrocapillary

29: dCTP injection electrocapillary

30: DNA synthase injection electrocapillary

31: Restriction enzyme injection electrocapillary                 

32: supply discharge module

33: needle for discharging the washing water

34: wash water reservoir

35: negative electrode for vertical transportation of supply discharge module

36: Positive electrode for vertical transportation of supply discharge module

37: Reaction material entrance on the side of the capillary

38: hydrophobic insulating layer of an electrocapillary tube

Currently, the main technique for analyzing DNA base sequence is based on the living method. In the Genome Project, the temperature is increased and decreased to use one template several times, repeated synthesis, electrophoresis in capillary tube, and DNA separated according to length. It is a living method that detects the location of fragments with a fluorescent dye labeled at the end of DNA and automatically recognizes the sequence. This method (Sanger Sequencing with cyclic sequencing and capillary electrophoresis) is time-consuming and expensive.

In addition to DNA sequencing, ddNTP with biotin instead of fluorescent dye was used for spectral spectrometry instead of electrophoresis. Pyrosequecing, Bulk-fluorescence DNA sequencing-by-synthesis using fluorescent dye dNTP to synthesize several DNA molecules, Single-molecule DNA sequencing using dNTP with fluorescent dye when synthesizing one DNA molecule, DNA Randomly shorten the molecules and hybridize them with specific fragments to find short sequences, and then computerized sequencing by hybridization that connects numerous overlapping sequences into one, and installs an extremely small nanopore in the lipid membrane between the two aqueous solutions. Nanopore DNA sequencing, which identifies DNA sequences by passing DNA molecules, attaches and removes specific fragments to DNA molecules. There are many ways to find the sequence, such as massively parallel sequencing with stepwise enzymatic ligation and cleavage. Techniques used in DNA chips (aka Gene chips) usually only reveal different DNA base sequences.

Among the existing methods, sequencing method using DNA synthesis causes many problems by attempting to perform long DNA synthesis and detection of consumed dNTPs in one reaction site. Micromechanical engineering and microfluidics continue to evolve as a way to identify long DNA sequences without electrophoresis by performing separate reactions to synthesize short DNA for detection separately in different places. DNA synthase, fluorescent dyes, and fluorescence detection equipment continue to develop, which can be improved to identify longer and longer DNA sequences.

dNTP depletion DNA synthesis reaction and dNTP depletion detection DNA synthesis reaction are performed in different places, and the consumption and degree of consumption of dNTP depletion detection DNA synthesis using simple methods without adversely affecting dNTP depletion DNA synthesis reaction. Can be seen.

The Sanger method is difficult to fundamentally improve the limitations and costs of electrophoretic analysis, and the other methods are still at the stage of confirming the principle, or at the level where short sequences can be identified due to various problems.

The present invention is a method to solve these problems, it is possible to economically identify long DNA sequences through the improvement of DNA synthase, the improvement of fluorescent dyes, the application of electrowetting phenomenon and the improvement of microfluidics.

Bulk-fluorescence DNA sequencing-by-synthesis, which uses DNA synthesis to identify sequences, uses dNTP analogs with fluorescent dyes to synthesize DNA. In this case, DNA polymerase attaches the first dNTP analog to DNA. Then there is a disadvantage that the synthesis efficiency falls sharply, but the present invention does not cause this problem because the normal dNTP is used in the DNA synthesis reaction for dNTP consumption or the DNA synthesis for dNTP consumption detection.

By using electrowetting phenomenon, biological buffers, enzymes, and DNA synthesis materials required for reaction can be supplied and washed at appropriate concentrations and amounts. Thus, the initiation, maintenance, termination, and washing of reactions can be precisely controlled. Before the dNTP consumption DNA synthesis reaction, the fluorescent dyes attached to the ends of the DNA primers were measured to measure the concentration of DNA molecules, supplying the appropriate concentration and quantity of material, and the DNA synthesis reaction for dNTP consumption detection. Measure the intensity of the fluorescent dyes attached to the ends of the DNA molecules before cutting them, cut them with restriction enzymes, wash them, and measure the change in the intensity of fluorescence. Then adjust the type and concentration of dNTPs supplied to the DNA synthesis for dNTP consumption. Base sequences can be determined even when they are in series.

When described in detail by the accompanying drawings as follows.

The present invention uses a reaction for synthesizing the DNA to determine the sequence and a separate DNA synthesis reaction to determine what dNTP was used in this synthesis.

If a specific dNTP has not been consumed in dNTP consuming DNA (DNA to identify base sequence) reaction, the specific dNTP depletion detection DNA synthesis reaction enters the fixed DNA, and then the remaining dNTPs that are waiting to enter are restricted to Sau3AI. GATC sequence synthesized by enzyme When DNA synthase finishes synthesis and Sau3AⅠ restriction enzyme cuts it, it uses the principle that the fluorescent dye attached to the end of DNA is lost when washing and cannot detect fluorescence.

When the DNA is prepared, the concentration is prepared. Before the DNA synthesis reaction for dNTP consumption (Fig. 1A), the fluorescence of Cy5 attached to the 5 'end is measured to increase the accuracy. After supplying dGTP and DNA synthetase to perform DNA synthesis reaction for dNTP consumption, and then sent to the reaction product DNA synthesis reaction for detecting dGTP consumption (Fig. 1B), there are only dATP, dTTP, and dCTP. Without the DNA synthesis reaction, dGTP is required for the initial synthesis in the detection DNA synthesis reaction. However, in the dNTP-consuming DNA synthesis reaction, 110% of the amount required for synthesis is supplied. If the first sequence is G, after the dNTP-consuming DNA synthesis reaction, 10% of dGTP remains in the reaction solution. About 10% of DNA was synthesized, and the synthesized DNA was cut off when Sau3AⅠ was added. In the DNA synthesis reaction for detection, the intensity of fluorescence was measured 90% of the measured fluorescence immediately before the restriction enzyme.

If the first nucleotide sequence is not G in the dNTP consuming DNA synthesis reaction, dGTP is not consumed and 100% of the DNA is synthesized in the dNTP consuming detection DNA synthesis reaction. In this case, a sequence recognized as the restriction enzyme Sau3AⅠ is generated to generate Sau3AⅠ. When added, the DNA is cleaved, and when washed, all are washed away and no fluorescence is detected.

100% of the fluorescence would be measured if the first base as well as the second base in the DNA synthesis for dNTP consumption. Therefore, if 100% of dGTP is supplied to the DNA synthesis reaction for dNTP consumption, 90% of the fluorescence will be measured if G is the second time, and 100% if the third degree is G.

Once 90% of the fluorescence was measured, dATP was fed to dNTP consuming DNA synthesis reaction.

dTTP is supplied when 90% of fluorescence is measured after synthesis, cleavage and washing in DNA synthesis reaction for dATP consumption detection. Supply dCTP in the same way.

By repeating this, it is possible to know which dNTPs have been consumed in the dNTP-consuming DNA synthesis reaction, so that the base sequence of the DNA synthesized in the dNTP-consuming DNA synthesis reaction can be known (FIG. 10A).

In the application drawing of the present invention, since each dNTP depletion detection DNA synthesis reaction site (12, 21, 22, 23 of Figure 4) is five lines, it can be confirmed up to the fifth nucleotide sequence, and if you increase it to thousands of lines, it is possible to identify up to thousands May be.

DNA synthesis reaction for dNTP depletion detection contains dNTPs after subtracting DNA synthetase and specific dNTP to be detected in synthetic reaction buffer, and DNA strands with fluorescence-labeled DNA primers are reacted through biotin and streptavidin. Stuck on. Restriction enzymes are supplied after DNA synthesis.

DNA synthesis reaction for detecting dGTP depletion contains dATP, dTTP, dCTP,

DNA synthesis reaction for dATP depletion detection contains dGTP, dTTP, dCTP,

DNA synthesis reaction for detecting dTTP consumption contains dGTP, dATP, dCTP,

DNA synthesis reaction for detecting dCTP depletion contains dGTP, dATP, and dTTP.

The primers are also attached to each other, so the dNTPs required for the first synthesis are different, and the sequence of DNA attached to the biotin is also different (Fig.

In the dNTP-consumed DNA synthesis reaction, when the reaction solution is sent to the dNTP depletion detection DNA synthesis reaction, the remaining solution of dNTP is washed with buffer because the reaction solution cannot be transferred 100% due to the hydrophilicity of the DNA and the attraction of the hydrophilic reaction site. smoking. At this time, some DNA synthase may be washed away, so the DNA synthase is supplemented separately from washing.

The buffer used for washing is pure DNA synthesis reaction buffer containing no enzymes or nucleotides.

Excessive supply of buffer to the DNA synthesis site for washing extends the boundaries of the liquid droplets to the outlet adjacent to it and draws liquid from the outlet (Fig. 1 or 13). By repeating this, the DNA strands with fluorescent dyes can be washed away.

Use an electrocapillary tube to inject buffers and other materials, and use a discharge needle using electrowetting to draw liquid from the outlet.

As a method of supplying the reactant material or the buffer solution, various methods of controlling the fine movement of the fluid may be used.

The orbit can be washed by transporting the wash buffer to wash away the dNTP, which may remain in trace amounts, while transporting the reaction solution to the orbit for transporting the reaction solution (FIG. 16A, FIG. 17A, B). Where the trajectory of the negative electrode is dispersed, the trajectory is doubled to solve the problem. Figure 18 (a) is the top and bottom of the negative electrode where the orbit is dispersed. (B) is seen from the side.

Buffers, dNTPs, DNA synthase and restriction enzyme reservoirs can be designed to be transported horizontally rather than vertically by installing them in the same plane as the DNA synthesis site.

Figure 1 (a) 10, (b) 12 is a hydrophilic surface for attaching biotin and streptavidin and a template DNA strand attached to the biotin may be made by dipping a hydrophilic material on the hydrophobic insulating layer or other various methods Can be used. In FIG. 4, 12, 21, 22, and 23 are not drawn in an exact proportion, but roughly show a circular interface of the droplet of the reaction solution, and the hydrophilic surface is smaller than that of the hydrophilic spot.

Transfer reaction buffer containing dNTP, DNA synthetase, and restriction enzyme using electrowetting phenomenon, and DNA synthesis reaction for detecting dNTP consumption after reaction of DNA synthesis in dNTP consumption DNA synthesis reaction And buffered large amounts to flush out the cut DNA after restriction enzyme reactions.

Transfer of liquid droplets using electrowetting is open and closed. 3 is a principle that the droplet is moved in the closed case.

Current flows only to the electrode in the direction of the drop.

The closed type was placed on top of a plate with a hydrophobic insulating layer (FIG. 1 to 3) and a transparent plate with a hydrophobic insulating layer thereon as well. The lower plate has the orbit of the cathode, and the site for the DNA synthesis for dNTP consumption and the DNA synthesis for the detection of dNTP consumption is a small circle with a hydrophilic surface, and streptavidin bound to biotin and biotin. has exist.

The upper plate has a transparent positive electrode (Indium Tin Oxide electrode, less than 15nm thick).

A hydrophobic film is formed on the glass surface in contact with the droplet for electrowetting except for the point where DNA is attached. Hydrophobic coatings can be used to create hydrophobic surfaces or nano-sized small irregularities, and hydrophobic membranes can be coated to create superhydrophobicity surfaces. The hydrophobic insulating layer is made of Dupont's Teflon AF with thickness less than 20nm. In addition, one of several commercially available materials can be used, including Fluoroplymer xR, ICP fluoropolymer, AKD, Fluoroalkylsilane (FAS), Octadecyl dimethylchlorosilane, and FluoroPel. Insulating layer formation methods include spin, spray, evaporation, plasma etch, etc., and there is a difference in hydrophobic strength depending on the thickness and droplet composition. The thinner the hydrophobic insulating layer, the smaller the current can move the droplets, and the droplets move through the current through one of the droplets in the direction of movement among the (-) electrodes.

In the open type, multiple (+) and (-) currents flow under the hydrophobic insulation layer in parallel, and the droplets move by passing current through only the wires on one side of the liquid in the direction of movement.

The closed type and the open type have advantages and disadvantages, respectively, and are being developed in parallel.

When amorphous silicon receives light, its conductivity changes, so that only a part of the electrodes listed by using alternating current flows, so that when the droplets are moved using opto-electrowetting (OEW), which moves the droplets, It is possible to use a laser in the range that does not bleach or to shine the laser from below to avoid contact with the reaction solution.

There are many studies on how to separate droplets from large to small droplets, cutting large droplets into two, merging two droplets, or transporting them. At lower voltages, they are evolving to move smaller amounts faster. If the droplets are small, the two droplets can be combined enough to be mixed by diffusion to avoid stirring. By mixing the reaction droplets under different conditions, the pH, temperature, dissolved enzyme, DNA, and various substances can be quickly controlled. As a result, the reaction can be stopped or the rate of the reaction can be controlled, and the effect of washing off is obtained by successively combining and removing droplets having a high concentration of a specific substance and droplets that do not contain the substance.

The droplets can move above 30mm / sec, control current droplets individually by supplying current in an active matrix type, and allow the droplets to stand right next to where they will react beforehand. When several drops are placed in a line, they can be combined with the reaction solution and drawn out to the outlet (Fig. 1 or 13) to obtain a cleaning effect. The reaction solution droplets (Fig. 1-5) are 0.5 to 1.0 µl or less, and the electrode can also react at a current rate in units of μm or several tens of nm, and in the future, increase the velocity of the reaction solution droplets by improving the hydrophobic insulating layer. You can.

The washes are withdrawn through the wash water draining needle (Fig. 6 33) close to the hole next to the reaction site.

The method of finely controlling the smaller electrode orbit (FIG. 2B) may be applied to the present invention, such as an active matrix type design manufacturing method.

For DNA synthesis reaction, T7 Sequenase ^TM DNA Polymerase is used as a DNA synthase for both dNTP consumption and dNTP consumption detection.

Restriction enzymes were used in Sau3AⅠ, and there are 4 nucleotide sequences that do not overlap with GATC.

The streptavidin (9 of Figure 1) is connected to the DNA molecule (7 of Figure 1) to try to find the biotin attached sequence on the 5 'side. At the 3 'end there is a primer with Cy5 (absorption 649 nm, emission 670 nm) at the 5' end. DNA synthase attaches dNTP to 3'-OH of this primer to synthesize DNA.

When detecting DNA, fluorescent dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, TxR, R6G, FITC, DEAC, various Alexa Fluor dyes, OG, coumarine and rhodamine derivatives After attaching hapten such as DIG, BIO, DNP, TAMRA, attach antibody to fluorescent dye, attach it with CCD camera, detect it with CCD camera, analyze with computer, and supply dNTP, wash, and transport the reaction solution near the reaction site in advance. To automatically adjust things.

In the present invention, five DNA synthesis reactions are used to determine the DNA sequence, and a DNA synthesis reaction for dNTP consumption and a DNA synthesis reaction for dGTP, dATP, dTTP, and dCTP depletion are separately performed. Will. The four dNTP depletion detection reactions can be performed in the following ways.

If a particular dNTP has been consumed in a consumable DNA synthesis reaction, it cannot be used for DNA synthesis in a depletion DNA synthesis reaction, and then dNTP-Cy5 (fluorophore-modified nucleotides) cannot be used for synthesis. Without specific dNTP, dNTP analogs with fluorescent dyes (Cy5) also cannot be used for synthesis, so their fluorescence is not detected after washing, and thus the sequence can be determined (Fig. 11A).

If a specific dNTP has been consumed in a consumption DNA synthesis reaction, it cannot be used for DNA synthesis in a consumption detection DNA synthesis reaction, and then aa-dNTP (aminoallyl-modified nucleotide) cannot be used for synthesis. After adding and washing amino-coupling of fluorescent dyes that react with the amine of the dNTP analog, the base sequence can be determined because the fluorescent dye is not detected (FIG. 11 b).

If a specific dNTP is consumed in the consumption DNA synthesis reaction, the DNA synthesis does not occur in the consumption detection DNA synthesis reaction, and the DNA fragment containing the fluorescent dye complementary to the nucleotide sequence of the template DNA strand may hybridize. Since fluorescence is detected, the sequence can be identified (FIG. 12).

When specific dNTPs are consumed in the consumption DNA synthesis reaction, DNA synthesis does not occur in the consumption detection DNA synthesis reaction, and the ferrocene-containing DNA (ferrocene-modified DNA) fragment, which is complementary to the nucleotide sequence of the template DNA strand, may hybridize. Since the electrical characteristics can be measured by alternating current voltammetry (alternating current voltammetry) can determine the nucleotide sequence (Fig. 12b).

If a specific dNTP has not been consumed in the consumption DNA synthesis reaction, the ppi produced when the dNTP is used in the DNA synthesis reaction in the consumption detection DNA synthesis reaction is changed to ATP with APS (adenosine 5 'phosphosulfate) and ATP sulfurylase, Luciferase can be used to detect nucleotide sequences by detecting the light that is produced when luciferin is converted into oxyluciferin. In this case, dATPαS (deoxyadenosine alfa-thio triphosphate) is used instead of dATP in the consumption DNA synthesis reaction. DNA polymerase reacts well with dATPαS but not luciferase. DNA synthesis reaction for consumption detection is used only once, so it is not necessary to use apyrase as in pyrosequencing (Fig. 13A).                     

If a specific dNTP has been consumed in the consumption DNA synthesis reaction, it cannot be used for DNA synthesis in the consumption detection DNA synthesis reaction. Therefore, after the synthesis reaction, the reaction solution is sent to a fixed place where biotin is attached to the complementary DNA template for consumption detection. When fluorescence can be detected after washing when the base sequence can be identified (Fig. 13b).

If a specific dNTP has been consumed in the DNA synthesis for consumption, it cannot be used for DNA synthesis in the consumption-detection DNA synthesis reaction. Therefore, an enzyme (eg, horeseradish peroxidase) is added to the DNA having the same sequence to be synthesized in the dNTP consumption detection DNA synthesis reaction. Since the attached DNA strands can hybridize, the substrate (eg 5AP 5-aminosalicylic acid) can be detected after washing to detect the color change and confirm the synthesis (FIG. 14A).

When a specific dNTP has been consumed in a consumable DNA synthesis reaction, the enzyme DNA (eg horseradish peroxidase) attached to the end of a DNA long enough to provide flexibility that does not interfere with DNA synthase activity. This DNA strand cannot be cut by restriction enzyme because it can't be used for DNA synthesis in the synthesis reaction. After washing, this enzyme is not lost and the color of the enzyme is changed when the substrate (5AP; 5-aminosalicylic acid, etc.) is supplied. By detecting this, the DNA sequence can be confirmed (FIG. 14 b).

If dNTPs were not consumed in the consumable DNA synthesis reaction, dNTPs enter the fixed DNA in the depletion DNA synthesis reaction, so that the next possible dNTPs will enter, and after they enter, they will be identical to the later sequence Since the DNA fragment containing the antibody is not hybridized, it is washed out when washing, and since the antibody is not attached when the antibody with fluorescent substance is added after washing, the sequence can be determined (FIG. 15).

In the DNA synthesis reaction for detecting consumption, if the antigen is attached to the 3 'side of the template DNA and can be cut into Sau3AⅠ, the nucleotide sequence may be identified because the antigen is lost when the antigen is washed and the antibody with the fluorescent substance cannot be attached.

The DNA attached to streptavidin-coated magnetic beads and the necessary DNA in one place close to the consumption DNA synthesis reaction are not used for dNTP depletion detection DNA synthesis reaction in different places. The sequence can also be determined by bringing the material to the magnetic needle site each time to react and rinse (FIG. 15B).

In hybridization detection, buffers can be supplied that are well-suited to detect the desired hybridization, and other reactions can be controlled appropriately by matching the appropriate conditions.

As described above, the present invention relates to a method for analyzing DNA sequencing. Among the existing methods, sequencing method using DNA synthesis is performed by performing long DNA synthesis and detection of consumed dNTP in one reaction site. There is a problem, and the present invention provides a method for synthesizing long DNA (dNTP consuming DNA synthesis) and short DNA to detect specific dNTP consumption (dNTP consuming DNA synthesis) in different places. Separately, the synthesis and detection reactions can be performed without such a problem, and thus a long DNA sequence can be identified without electrophoresis.

Claims (12)

When synthesizing DNA to identify the nucleotide sequence, a specific dNTP is given to perform DNA synthesis for dNTP consuming, and then the reaction solution is required for this specific dNTP to start the synthesis reaction to synthesize a nucleotide sequence recognized by restriction enzyme. DNA synthesis reactions were sent to the dNTP depletion detection DNA synthesis reaction, and then the restriction enzyme was added to participate in synthesizing the sequence recognized by the restriction enzyme due to the presence of this specific dNTP. Without this specific dNTP, the restriction enzyme could not synthesize a recognizable sequence and the DNA would not be cleaved when the restriction enzyme was added. If the DNA associated with the fluorescent material could not be washed away, this particular dNTP was used to synthesize the DNA to identify the sequence. DNA sequencing method using DNA synthesis that proceeds dNTP consumption DNA synthesis reaction and dNTP consumption detection DNA synthesis reaction in different places. As in claim 1, as a method of proceeding with dNTP consumption DNA synthesis reaction and dNTP consumption detection DNA synthesis reaction at different places, only dNTP consumption detection DNA synthesis reaction is different from the method of claim 1 in the present invention. Included. If a particular dNTP has been consumed in a consumable DNA synthesis reaction, it cannot be used for DNA synthesis in a depletion DNA synthesis reaction, and then dNTP-Cy5 (fluorophore-modified nucleotides) cannot be used for synthesis. If there is no specific dNTP, dNTP analogs with fluorescent dye (Cy5) also cannot be used for synthesis, so their fluorescence is not detected after washing. If a specific dNTP has been consumed in a consumable DNA synthesis reaction, it cannot be used for DNA synthesis in a depletion DNA synthesis reaction and then aa-dNTP (aminoallyl-modified nucleotide) cannot be used for synthesis. DNA sequencing method using DNA synthesis to determine the sequencing because the fluorescent dye is not detected after adding and washing amino-coupling of fluorescent dyes reacting with amines of dNTP analogs. If a specific dNTP is consumed in the consumption DNA synthesis reaction, DNA synthesis does not occur in the consumption detection DNA synthesis reaction, and the DNA fragment containing the fluorescent dye complementary to the nucleotide sequence of the template DNA strand may hybridize. DNA sequencing method using DNA synthesis to identify the nucleotide sequence because fluorescence is detected. When specific dNTPs are consumed in the consumption DNA synthesis reaction, DNA synthesis does not occur in the consumption detection DNA synthesis reaction, and the ferrocene-modified DNA fragment containing DNA complementary to the nucleotide sequence of the template DNA strand can be hybridized. DNA sequence analysis method using DNA synthesis to determine the sequence by measuring alternating current voltammetry (electrical characteristics) can be different. If a specific dNTP was not consumed in the DNA synthesis for consumption, the ppi generated when the dNTP was used in the DNA synthesis for consumption detection was changed to ATP with APS (adenosine 5 'phosphosulfate) and ATP sulfurylase. DNA sequencing method using DNA synthesis to detect the light sequence generated when luciferin is made into oxyluciferin with luciferase. If a specific dNTP has been consumed in the consumption DNA synthesis reaction, it cannot be used for DNA synthesis in the consumption detection DNA synthesis reaction. After the synthesis reaction, the reaction solution is sent to a fixed place where biotin is attached to the complementary DNA template for consumption detection. DNA sequencing method using DNA synthesis to grasp the nucleotide sequence because it can detect the fluorescence after washing. If a specific dNTP has been consumed in the DNA synthesis for consumption, it cannot be used for DNA synthesis in the consumption-detection DNA synthesis reaction. Therefore, an enzyme (eg, horeseradish peroxidase) is added to the DNA having the same sequence to be synthesized in the dNTP consumption detection DNA synthesis reaction. DNA sequencing method using DNA synthesis to identify the nucleotide sequence by detecting the change of color by giving a substrate (eg 5AP 5-aminosalicylic acid) after washing, since the attached DNA strands can hybridize. When a specific dNTP has been consumed in a consumable DNA synthesis reaction, the enzyme DNA (eg horseradish peroxidase) attached to the end of a DNA long enough to provide flexibility that does not interfere with DNA synthase activity. This DNA strand cannot be cut by restriction enzyme because it can't be used for DNA synthesis in the synthesis reaction. After washing, this enzyme is not lost and the color of the enzyme is changed when the substrate (5AP; 5-aminosalicylic acid, etc.) is supplied. DNA sequencing method using DNA synthesis to detect the DNA sequence by detecting this. If dNTPs were not consumed in the consumable DNA synthesis reaction, dNTPs enter the fixed DNA in the depletion DNA synthesis reaction, so that the next possible dNTPs will enter, and after they enter, they will be identical to the later sequence Since the DNA fragment containing the antibody cannot hybridize, it is washed out when washing, and when the antibody with fluorescent substance is added after washing, the antibody does not adhere to it. Once the antigen is attached and can be cut with Sau3AⅠ, the antigen is lost when washing and the antibody attached with the fluorescent substance cannot attach to the DNA sequencing method using DNA synthesis to identify the sequence. The DNA attached to streptavidin-coated magnetic beads and the necessary DNA in one place close to the consumption DNA synthesis reaction are not used for dNTP depletion detection DNA synthesis reaction in different places. DNA sequencing method using DNA synthesis to grasp the nucleotide sequence by bringing the material to the magnet needle place every time to react and wash.

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