JP2009011230A - Method for analyzing base sequence by utilizing restricted extension of nucleotide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for efficiently carrying out analysis of a desired region (e.g. a coding region on a mRNA and a cDNA, in which a poly(A) region is intervened between a primer-binding site and a sequence information-required region) which is remote from a primer-binding site on a template nucleic acid, without having to make a specific primer in a sequence analysis by a sequence-analysis method that uses a monomolecular polynucleotide chain as a template, a pyrosequencing method, and the like. <P>SOLUTION: The method for analyzing a base sequence by utilizing a DNA extension reaction is carried out, after previously extending a primer from the reaction system to this side of a deleted base by carrying out DNA extension reaction, in a state in which one among four kinds of nucleotides of dATP, dCTP, dGTP and dTTP is deleted, and repeating the reaction by changing the kind of the deleted base before the sequence analysis. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a nucleotide sequence analysis method. More specifically, the present invention relates to a base sequence analysis method for sequencing a region away from a primer binding site on a target nucleic acid by a nucleic acid analysis method using a base extension reaction such as single molecule sequencing or pyrosequencing.

  The DNA and RNA base sequencing method is an important technique. Usually, a sample in which a DNA fragment or a group of fragments for sequencing is preliminarily labeled with a fluorescent label is prepared, and analyzed by measuring a molecular weight separation development pattern after electrophoresis or during electrophoresis. Specifically, prior to electrophoretic separation, a dideoxy reaction by a well-known Sanger method is performed. An oligonucleotide having a length of about 20 bases complementary to the known base sequence portion of the sample DNA to be analyzed is synthesized and the fluorescent substance is labeled. Using this oligonucleotide as a primer, complementary strand binding is carried out with about 1 pmol of sample DNA, and a complementary strand extension reaction is carried out by polymerase. At this time, four types of deoxynucleotide triphosphates, that is, dATP, dCTP, dGTP, dTTP, and in addition to these, for example, ddATP are added as substrates. When ddATP is incorporated by complementary strand extension, the complementary strand does not extend any more, so fragments of various lengths terminating in adenine (A) are prepared.

  In the above reaction, ddCTP, ddGTP, and ddTTP are added in place of ddATP. However, the primers used in each reaction have the same base sequence, but are labeled with four types of phosphors that can be distinguished from each other by spectroscopy of fluorescence. When the above four types of reactants are mixed, fragments that differ in length by one base up to several hundred bases complementary to the sample DNA are labeled with four different phosphors depending on the terminal base type. can get. This is separated by capillary gel electrophoresis with a resolution of 1 base. Samples are migrated while being separated, and laser irradiation is performed in order from the shortest. When the emission fluorescence is measured using a plurality of filters while spectroscopy, the terminal base types of all the fragments can be determined in order from the shortest fragment based on the temporal change in the fluorescence intensity of the four phosphors. In addition, you may use labeled dNTP which each labeled ddATP-ddTTP (ddNTP) with a different fluorescent substance, without fluorescently labeling a primer.

  In recent years, a pyrosequencing method that utilizes the release of pyrophosphate in association with a DNA extension reaction has been put to practical use as a method for acquiring sequence data at the same time as the reaction proceeds using a base extension reaction. Patent Document 1). In this method, sample DNA is immobilized on a bead or a substrate, a specific base is supplied to the sample DNA from four dNTPs, the presence or absence of an extension reaction is detected by the presence or absence of release of pyrophosphate, and the sequence of the sample DNA is determined. I will decide. Specifically, ATP is produced from the released pyrophosphate and adenosine 5′-phosphosulfate by the catalytic reaction of ATP sulfurylase. Since this ATP causes a luminescence reaction in the presence of luciferin and luciferase, the presence or absence of pyrophosphate release is confirmed by the presence or absence of luminescence. Furthermore, a method has also been proposed in which a sample DNA fragment to be analyzed randomly is captured on the substrate surface one molecule at a time, extended by approximately one base, and the result is detected by fluorescence measurement to determine the base sequence. Specifically, as a substrate for DNA polymerase, four dNTP derivatives (MdNTPs) having a detectable label that can stop the DNA chain extension reaction due to the presence of a protecting group when incorporated into the template DNA are used. The step of performing a DNA polymerase reaction, the step of detecting the incorporated MdNTP by fluorescence or the like, and the step of returning the MdNTP to an extendable state are taken as one cycle, and the base sequence of the sample DNA is determined by repeating this cycle. . In this technique, since DNA fragments can be sequenced one molecule at a time, many fragments can be analyzed at the same time, and the analysis throughput can be increased.

  In any of the above methods, a specific primer is annealed to the sample DNA, and an extension reaction is caused from the 3 'end of the primer. That is, the sequence analysis is started from the position where the primer is hybridized.

  One of the purposes of base sequence analysis is to analyze the sequence of messenger RNA (mRNA) (poly (A) + RNA) expressed in cells and to analyze genes expressed in cells. When identifying genes from sequence analysis results by homology search using a sequence database, it is possible to identify 3 'downstream non-coding region sequences that are not polyadenine, but all or part of the amino acid sequence coding region. It is most desirable that the following sequence is obtained. However, since the length of the polyadenine portion and the non-coding region at the 3 ′ end side of poly (A) + RNA varies depending on the poly (A) + RNA molecule, the poly (A ) + RNA molecules may not be identified. Although long base length sequence reading is desirable, it may be difficult in principle due to equipment, reaction, etc.

  Therefore, a method for obtaining information such as an amino acid coding region or a 3 'untranslated region other than the poly (A) portion with a short base sequence length is desired.

Analytical Biochemistry, vol. 242 (1), pp84, 1996 Proc. Natl. Acad. Sci. USA, vol. 100 (7), pp3960, 2003

  In a sequence analysis method using a base extension reaction such as a sequence analysis method using a single molecule polynucleotide chain as a template or a pyrosequencing method, a sequence analysis of poly (A) + RNA is performed using an oligo (dT) primer Has the following difficulties. The short-length oligo dT does not uniquely determine which part of the poly (A) sequence hybridizes, and hybridizes randomly. Therefore, in the sequence analysis method using a single-molecule polynucleotide chain as a template, it is difficult to obtain a substantially necessary sequence when the base length to be read is short because the polyadenine moiety is interposed between the primer and the originally required sequence information. There are many cases. On the other hand, when a sample (or primer) is immobilized on a solid phase such as a pyrosequencing method, usually, the same target nucleic acid is immobilized at the same position, and when oligo (dT) is hybridized to them, for the reasons described above, Hybridization at different positions with respect to the same target nucleic acid. It is difficult to place all hybrids at the same position on poly (A). Therefore, even if each base is extended, the sequence position is different, resulting in a mixed signal, making sequencing difficult. Therefore, a method for synchronizing is desired.

  In the above case, the present invention provides a method for sequence analysis of a necessary region without preparing a new primer, and a sequence analysis apparatus using the method.

  In response to the above problems, the inventors performed a DNA extension reaction in the state lacking at least one of the four nucleotides dATP, dCTP, dGTP, and dTTP prior to the sequencing reaction, and lost the reaction system. It was found that by repeating the step of extending the primer to the front of the base, while changing the type of the defective base, the sequence analysis can be started from a portion that has greatly advanced from the hybridized position of the primer.

That is, the present invention provides a base sequence analysis method including the following steps:
1) Hybridize the primer to the nucleic acid to be analyzed;
2) The nucleic acid to be analyzed is subjected to an extension reaction with DNA polymerase in a reaction system lacking at least one base of dATP, dCTP, dGTP, and dTTP;
3) After the extension reaction, the DNA polymerase and the base are removed from the reaction system, and a sequence analysis reaction using the nucleic acid extension reaction is performed to determine a desired region away from the primer binding site on the nucleic acid to be analyzed. To do. “A desired region apart from the primer binding site” is a region requiring sequence information 5 ′ upstream from the primer binding site ”, for example, when a repetitive sequence is in the vicinity of the primer binding site. It is possible to determine the sequence from the area that requires the sequence information by skipping that portion. Although it depends on the sequence of the target nucleic acid, the analysis position of several bases to several tens of bases can be shifted.

  In one embodiment, the steps 2) and 3) are performed a plurality of times while changing the type of the defective base.

  In one embodiment, the nucleic acid to be analyzed is mRNA having a poly (A) region or cDNA derived from mRNA, and the primer is an oligo (dT) primer.

  In one embodiment, the analysis target is mRNA having a poly (A) region, the primer is an oligo (dT) primer, and an extension reaction is performed in a reaction system lacking at least one base other than dTTP. Do.

  In one embodiment, the analysis target is mRNA derived from cDNA having a poly (T) region, the primer is an oligo (dA) primer, and the reaction lacks at least one base other than dATP. Perform an elongation reaction in the system.

  In the above embodiment, the extension reaction is performed a plurality of times while changing the type of the defective base.

  The base sequence determination method of the present invention can be used to determine the sequence of a desired region without amplifying the nucleic acid to be analyzed, and thus is useful in genome analysis and the like that require analysis of a small amount of nucleic acid.

As one embodiment of the base sequence determination method of the present invention, a base sequence analysis method including the following steps can be mentioned:
1) Hybridizing oligo (dA) primer to cDNA derived from mRNA and having a poly (T) region;
2) An extension reaction is performed by adding DNA polymerase and dATP to the cDNA;
3) After the extension reaction, DNA polymerase and dATP are removed from the reaction system;
4) Sequencing is performed by conducting a sequence analysis reaction utilizing a nucleic acid extension reaction.

Another embodiment of the base sequence determination method of the present invention includes a base sequence analysis method including the following steps:
A nucleotide sequence analysis method comprising the following steps:
1) Hybridize oligo (dT) primer to mRNA with poly (A) region;
2) An RNA-dependent DNA polymerase and dTTP are added to the mRNA to perform an extension reaction;
3) After the extension reaction, RNA-dependent DNA polymerase and dTTP are removed from the reaction system;
4) Sequencing is performed by conducting a sequence analysis reaction utilizing a nucleic acid extension reaction.

The present invention also provides an apparatus for carrying out the above-described base sequence analysis method. This device:
A chamber for performing an extension reaction from the primer of the nucleic acid to be analyzed and a sequence analysis reaction;
A container and storage unit for storing the four substrate bases and reagents necessary for the reaction;
A reagent supply system for supplying the substrate base and a reagent required for analysis to the chamber in any combination in a state lacking at least one base;
A heating and cooling system for controlling the temperature in the chamber;
A detection system for detecting the sequence analysis reaction;
And an analysis system for analyzing the detection result.

  The apparatus may further include a container for storing four types of labeled dNTPs depending on the base sequence analysis reaction to be used. The dNTP derivative or label is not particularly limited, and examples thereof include dNTP and ddNTP having a known protective group, fluorescent label, radiolabel, and enzyme label.

The apparatus of the present invention may use a substrate base mixed reagent in a state lacking at least one base. In that case, the device:
A chamber for performing an extension reaction from the primer of the nucleic acid to be analyzed and a sequence analysis reaction;
A container and storage unit for storing one or more substrate base mixed reagents lacking at least one base, reagents necessary for the reaction, and four dNTPs or four labeled dNTPs, respectively;
A reagent supply system for supplying the reagent in the storage unit to the chamber;
A heating and cooling system for controlling the temperature in the chamber;
A detection system for detecting the sequence analysis reaction;
And an analysis system for analyzing the detection result.

  According to the present invention, primer binding on a template nucleic acid can be performed without producing a special primer in sequence analysis using a base extension reaction such as sequence analysis using a single molecule polynucleotide chain as a template or pyrosequencing. Analysis of a desired area away from the site can be performed. As a result, it is possible to efficiently analyze mRNA and cDNA in which the poly (A) region is interposed between the region requiring sequence information and the primer binding site.

  In the method of the present invention, in the first step, a nucleic acid molecule and a primer having a sequence complementary to a part of the sequence of the nucleic acid molecule are hybridized in a buffer in which a normal DNA polymerase reaction is performed.

One of the nucleic acid molecule and the primer may be immobilized on the support surface in advance. Further, both of them may be immobilized as long as they are immobilized after hybridization.
In the second step, a solution containing DNA polymerase and a base lacking at least one of dATP, dCTP, dGTP, and dTTP is provided to the hybridized nucleic acid molecule and primer, and a base is added to the 3 ′ end of the primer. React.

  In the third step, after the reaction, a reaction solution containing at least a base is separated from the nucleic acid molecule. When the nucleic acid molecule is immobilized on the surface of the carrier, it is washed away with at least a buffer solution containing no base. If not immobilized on a carrier, the nucleic acid molecule is recovered by ethanol precipitation.

  Thereafter, the second step and the third step are repeated as many times as necessary. In this case, the type of nucleotide not added to the reaction is changed every time. For example, dATP, dCTP, dTTP is added to the reaction in the first cycle, and dATP, dGTP, dTTP is added to the reaction in the next cycle. The number of repetitions is not particularly limited, and is appropriately determined according to a region where analysis target nucleic acid or base sequence information is required. When the analysis target is mRNA or mRNA-derived cDNA, depending on the sequence, it is usually performed 1 to 3 times.

  In the fourth step, the target nucleic acid molecule to be analyzed is subjected to a sequence analysis reaction using a nucleic acid extension reaction. The sequence analysis reaction using the nucleic acid extension reaction may be any known method, and can be applied to, for example, the sequence bi-sense method, the Sanger method, the pyro sequence method, and the like.

  Hereinafter, the method of the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  In the following examples, the reagent corresponding to the base T can be appropriately changed to U depending on the target nucleic acid (RNA or DNA).

  An artificially designed template DNA was used. The sequence of the oligo DNA used for the template DNA (SEQ ID NO: 1) and the primer (SEQ ID NO: 2) is as described in the sequence listing. The 5 'end of the primer is labeled with biotin.

0.1 pmole oligo (dT) primer is mixed with 10 μL of streptavidin-conjugated beads to immobilize the oligo (dT) primer on the beads. After washing with 100 μL each of washing buffer (10 mM TrisHCl pH 7.5, 7 mM MgCl ₂ , 50 mM NaCl) 3 times, add template DNA equal to oligo (dT) primer, stir well, heat at 75 ° C. for 10 minutes, then cool To do. After washing with 100 μL each of the washing buffer three times, a reaction buffer containing 5 pmole of dTTP and 0.5 unit of E. coli DNA polymerase Klenow fragment is added and reacted at 37 ° C. for 10 minutes. Further, after washing with 100 μL each three times with a washing buffer, a reaction buffer (10 mM TrisHCl pH 7.5, 7 mM MgCl ₂ , 0.1 mM DTT) containing 5 pmole of Cy3-dCTP and 0.5 unit of E. coli DNA polymerase Klenow fragment was added, and 37 Incubate for 10 minutes. Next, the fluorescence intensity of Cy3 is measured, and the sequence is determined.

  By the above procedure, the 5 ′ upstream sequence of the poly (A) sequence can be analyzed in sequence analysis using a fluorescent dye-labeled base.

  The above process will be described with reference to FIG. The oligo (dT) primer hybridizes to any position in the poly (A) region. In the example, 1a-c is hybridized to the center of the poly (A) region, and 2a-c is hybridized to the 5 'end of the poly (A) region. The hybrid position is determined probabilistically, and the hybrid state of the primer and template immobilized on the beads by the above reaction is a mixture of 1a, 2a and others. When dTTP and DNA polymerase are added to these, an extension reaction occurs in 1a, and the 3 'end of the primer reaches the 5' end of the poly (A) region, but the reaction stops there (1b). On the other hand, no elongation reaction occurs in 2a (2b). After this, when Cy3-dCTP and DNA polymerase are added, an elongation reaction occurs similarly in 1b and 2b, and G (guanine) can be determined as the next base after the end of the poly (A) region.

  FIG. 3 and FIG. 4 are fluorescence intensities showing the stretched state obtained based on the method of the present embodiment and their explanatory schematic diagrams. Condition (a) is a reaction with Cy3-dCTP after an extension reaction with dTTP, Condition (b) is a reaction with Cy3-dCTP without a dTTP reaction, Condition (c) (d) is a comparison Condition (c) is a sample, in which Cy3-dGTP is reacted without performing dTTP reaction. Condition (d) is the case of only beads that do nothing, and the fluorescence intensity obtained at that time is shown in FIG. . The sample solution is washed after the above reaction to remove the floating phosphor and the like and then redispersed in the solution. In the fluorescence measurement, the re-dispersed sample solution is put into a micro-quartz cell for fluorescence measurement, and the excitation wavelength is about 530 nm (any wavelength that can excite Cy3) and the fluorescence detection wavelength is about 580 nm (with a normal fluorescence spectrometer). The excitation wavelength is cut and any wavelength that can detect fluorescence is set). Under the condition (a), a large fluorescence intensity is obtained, but under the condition (b), the fluorescence intensity is small, and under the conditions (c) and (d), an intensity comparable to the background light is obtained. The primer and the poly (A) region of the template hybridize at an arbitrary position, but under the condition (a), almost all hybridized primers extend to the end of the poly (A) region, as in 1b and 2b of FIG. , Cy3-dCTP is considered to be bound (FIG. 4—condition (a)), and is considered to exhibit high fluorescence intensity. On the other hand, under condition (b), Cy3-dCTP binds only to the hybridized primer in the same state as 2a in FIG. 1 (FIG. 4—condition (b)), so only weak fluorescence is obtained (FIG. 1). There are only a few stochastic things in the same state as 2a). Under conditions (c) and (d), Cy3-dCTP does not expand as in FIG. 4 -conditions (c) and (d), so the intensity is about the same as background light. In FIG. 4, the solid phase and the primer are displayed so that the lengths are different from each other, but in actuality, it is assumed that they are immobilized in the same state.

  According to this example, in the case of a template nucleic acid in which the next base of the poly (A) region end point is G as in this example, dTTP is hybridized after oligo (dT) primer and before extension reaction with Cy3-dCTP. By performing a limited extension reaction using a polymerase enzyme, dCTP can be detected accurately and accurately as compared to the normal state in which no limited extension reaction is performed.

  In this example, the base sequence next to the end point of the poly (A) region is not limited to G, and C and T can be used to determine the base sequence accurately using Cy3-dGTP and Cy3-dATP. Further, the phosphor is not limited to Cy3, and any known phosphor such as Cy5 or rhodamine can be similarly reacted and carried out.

  In this example, a synthetic nucleic acid is used as the template nucleic acid, but it can also be applied to mRNA having a poly (A) region having the same structure, and the base can be accurately determined. In this case, as is well known, a polymerase having RNA activity is used.

  Further, the same measurement can be performed in the case of cDNA derived from mRNA. Since the cDNA in this case has a poly (T) region, the oligo (dA) primer is used instead of the oligo (dT) primer in the above example, and a limited extension reaction is performed using dATP and a polymerase enzyme. As a result, dCTP can be detected accurately and accurately compared to a normal state in which no restriction extension reaction is performed.

  In addition to the above examples, the fluorescence measurement can be performed using an apparatus capable of measuring various fluorescence intensities such as a fluorescence microscope.

  Moreover, although the system which fixed the primer to the bead (solid phase) was demonstrated, it can carry out similarly by fixing the nucleic acid which is a measuring object to a solid phase, and adding a primer and making it react.

  It can also be realized by using a substrate such as a glass slide in addition to beads as a solid phase.

  Moreover, the sequence analysis position on the same bead can be aligned by performing the extension reaction restricted beforehand. For example, it can be aligned with the end of the poly A region, and when multiple identical nucleic acids are captured on a bead solid phase, an elongation reaction occurs at the same position of almost all nucleic acid molecules, enabling highly accurate measurement. Become. When the extension reaction limited in advance is not performed, the position for sequence analysis differs for each nucleic acid molecule, so that an accurate sequence cannot be determined.

  A system using a glass flat plate as a solid phase will be described.

  The template DNA, primer DNA, and washing buffer are the same as in Example 1. FIG. 5 shows a schematic diagram in the reaction process.

Two pieces of double-sided tape cut to about 5 x 20 mm are pasted in parallel at a distance of about 15 mm on a thoroughly cleaned glass slide. An 18 × 18 mm cover glass is bonded onto this double-sided tape to form a reaction chamber. Add an aqueous solution of Poly-L-Lysine (containing 10% Poly-L-Lysine) to a 30 μL reaction chamber and let stand for 1 minute. Remove the Poly-L-Lysine aqueous solution by aspirating with filter paper, and wash the chamber by aspirating 3 times with 30 μL of water each time. Thereafter, an aqueous solution containing 1% biotinylated BSA is added to a 30 μL reaction chamber and left for 5 minutes. As in the above operation, remove the aqueous solution with filter paper and wash the chamber three times with 30 μL of water each time. Next, an aqueous solution containing 1% of streptavidin is added to a 30 μL reaction chamber and left for 5 minutes. Remove the aqueous solution with filter paper, and wash the chamber three times with 30 μL of water each time. An aqueous solution containing DNA hybridized in advance with the same amount of template DNA as 0.1 pmole oligo (dT) primer is added to a 30 μL reaction chamber and left for 5 minutes (FIG. 5A). Washing buffer (10mM Tris-HCl pH 7.5, 7mM MgCl 2, 50mM NaCl) After washing 3 times each 30μL in reaction buffer (10mM Tris-HCl pH containing E. coli DNA polymerase Klenow fragment of dTTP and 0.5 units of 0.5pmole 7.5, 7 mM MgCl ₂ , 0.1 mM DTT) is added to the chamber and allowed to react for 15 minutes at room temperature (FIG. 5B). After further washing three times with wash buffer each 30 [mu] L, reaction buffer containing E. coli DNA polymerase Klenow fragment of Cy3-dCTP and 0.5 units of 0.5 pmole (10mM TrisHCl pH 7.5, 7mM MgCl 2, 0.1mM DTT) was added, The reaction is allowed to proceed at room temperature for 15 minutes (FIG. 5 (c)). Next, fluorescence is observed under a microscope using total reflected light, and sequencing is performed. FIG. 6 shows a configuration diagram of the measuring apparatus, and FIG.

  FIG. 8 is an example of a captured image of the high sensitivity two-dimensional CCD camera of FIG. 6 obtained in this embodiment. The numbers and arrows in the figure show some of the fluorescent spots that occur, but a plurality of such bright spots can be detected as shown in the figure. The measurement is performed by irradiating the glass substrate surface with a laser beam having a wavelength of 532 nm at an appropriate angle so as to satisfy the total reflection condition on the upper surface of the glass. Thereby, an evanescent field is generated on the upper surface of the glass substrate, which excites Cy3-dCTP and emits fluorescence. Since the oligo (dT) primer is randomly fixed on the substrate, the fluorescent bright spots from Cy3 are also randomly observed as shown. This bright spot is the fluorescence from the Cy3-dCTP1 molecule, that is, indicates the elongation of each molecule of the nucleic acid to be measured, and the base sequence can be determined.

  In the case where a restriction extension reaction using dTTP and a polymerase enzyme is not performed after the oligo (dT) primer hybridization and before the extension reaction with Cy3-dCTP, as in the case of Example 1, Cy3 Even when -dCTP is added and reacted, Cy3-dCTP does not bind to the target nucleic acid, and the fluorescence emission points decrease. Probabilistically, fluorescence is emitted only in the case of the nucleic acid molecule hybridized to the state 2a in FIG. 1, but the fluorescence of most nucleic acid molecules cannot be obtained and the sequence cannot be determined. That is, the base sequences of many target nucleic acids can be determined by performing a limited extension reaction using dTTP and a polymerase enzyme.

  In this example, the base sequence next to the end point of the poly (A) region is not limited to G, and C and T can be similarly determined using Cy3-dGTP and Cy3-dATP. Further, the phosphor is not limited to Cy3, and any known phosphor such as Cy5 or rhodamine can be similarly reacted and carried out.

  In this example, a synthetic nucleic acid is used as the template nucleic acid, but it can also be applied to mRNA having a poly (A) region having the same structure, and the base can be accurately determined. In this case, as is well known, a polymerase having RNA activity is used.

  Further, the same measurement can be performed in the case of cDNA derived from mRNA. Since the cDNA in this case has a poly (T) region, the oligo (dA) primer is used instead of the oligo (dT) primer in the above example, and a limited extension reaction is performed using dATP and a polymerase enzyme. Thus, the bases of many nucleic acid molecules can be determined as compared with the normal state in which no restriction extension reaction is performed.

  In the above example, the case of determining the sequence of one base has been described. However, after performing a limited extension reaction using dTTP and a polymerase enzyme, Cy3-dATP, Cy3-dCTP, Cy3-dGTP, Cy3 By reacting with -dTTP, Cy3-dATP, Cy3-dCTP, Cy3-dGTP, Cy3-dTTP,... and detecting them, the base sequence can be determined continuously. In this case, each time the type of the base is changed, it is necessary to clean the substrate, bleach the phosphor, or desorb the phosphor molecule part. Moreover, what is labeled with a different phosphor for each base type may be used, and four types of phosphor-labeled bases may be mixed and reacted, and the fluorescence measurement may be detected by separating the four colors. In this case, in one reaction, it can be determined which base type is one base of the target nucleic acid, and rapid sequence analysis becomes possible.

  When four kinds of bases are reacted in order, if a limited extension reaction is performed using dTTP and a polymerase enzyme, it can be determined as “GCGCGGGTGA...” From the first base of detection. On the other hand, when the restriction extension reaction is not performed in advance, various patterns exist in the hybrid state. For example, “AAAAAAAAAG...” Is detected, and the meaningless poly A portion is mainly detected. In particular, when the sequencing base length is short, for example, if it is 10 bases, the case where the restriction extension reaction is not performed in advance, the effective base length is shortened, or it may be impossible to read at all. If the restriction extension reaction is performed in advance, detection of the poly A moiety can be automatically skipped, and the base sequence can be determined effectively.

  By the above procedure, the 5 ′ upstream sequence of the poly (A) sequence can be analyzed in the base sequence analysis of single-molecule DNA using a fluorescent dye-labeled base.

  The template DNA, primer DNA, and washing buffer are the same as in Example 1.

0.1 pmole oligo (dT) primer is mixed with 10 μL of streptavidin-conjugated beads to immobilize the oligo (dT) primer on the beads. After washing 3 times with 100 μL each with washing buffer, add the same amount of template DNA as the primer, stir well, heat at 75 ° C. for 10 minutes, and then cool. After washing 3 times with the washing buffer, a reaction buffer containing 5 pmole dTTP and 0.5 unit of E. coli DNA polymerase Klenow fragment is added to the beads and reacted at 37 ° C. for 10 minutes. After washing with 100 μL each 3 times with the washing buffer, dCTP (6.5 μM)), E. coli DNA polymerase Klenow fragment (1.0 unit / mL), ATP sulfurylase (0.3 unit / mL)), adenosine 5′-phosphosulfate (2.5 μM) ), Luciferase (20 μg / mL), luciferin (300 μM)) containing a reaction buffer (10 mM Tris-HCl pH 7.5, 7 mM MgCl ₂ , 1 mM DTT, 0.1% Tween 20) is added. Luminescence is measured with a plate reader while reacting at 37 ° C. for 10 minutes to determine the sequence.

  By the above procedure, the 5 ′ upstream sequence of the poly (A) sequence can be analyzed using the pyrosequencing method.

  FIG. 2 shows an apparatus for carrying out the method of the present invention. After the analysis target is immobilized on the reaction substrate 8, it is attached to the flow chamber 9 and the reaction is performed. The temperature of the reaction is controlled by a heating / cooling device of 7. Reagents and buffers are stored in 3 reagent storage units, mixed in 4 dispensing units, and sent to the reaction tank through 5 liquid feeding tubes and 6 reagent inlets. The reagent after the reaction accumulates in 11 waste liquid containers through 10 waste liquid tubes. Data of sequence analysis reaction is taken into 14 control PCs through 12 two-dimensional sensor cameras attached above the reaction tank, and can be observed with 15 monitors. Reagent feeding and reactor temperature are controlled through the control PC. In the figure, an apparatus using the pyrosequencing method is shown. However, if a container for storing four types of dNTP fluorescent derivatives is added to the three reagent storage units, a sequence analysis apparatus using the fluorescent derivatives can be obtained. In addition, if the signal is detected with a two-dimensional sensor camera through a microscope, an apparatus for sequence analysis using a single molecule polynucleotide chain as a template is obtained.

  The template DNA, primer DNA, and washing buffer used in the following sequence analysis reaction are the same as those in Example 1.

  The oligo (dT) primer is mixed with streptavidin-conjugated beads and the oligo (dT) primer is immobilized on the beads. After washing three times with the washing buffer, add the same amount of template DNA as the primer, stir well, heat at 75 ° C. for 10 minutes, and then cool. The beads on which the complex of primer and template DNA is immobilized are dispersed and immobilized on the reaction substrate 8 whose surface is biotinylated. A reaction substrate is mounted in the flow chamber 9 of the array analyzer. Wash with washing buffer held in 3 g of container. The dTTP held in the container 3d and the DNA polymerase reaction solution held in the container 3e are mixed and sent to the flow chamber 9 and reacted for 10 minutes while warming to 37 ° C. After the reaction, it is washed with a washing buffer held in 3 g of the container. Then, the dATP held in the container 3a, the DNA polymerase held in the container 3f, and the luminescence reaction solution are mixed and sent to the flow chamber 9 and heated to 37 ° C., and the luminescence signal is detected by the two-dimensional sensor camera 12. To do. Then wash again with wash buffer. Further, the same reaction, detection and washing are performed for dCTP, dGTP and dTTP. This is one cycle of reaction, and the base sequence is determined by repeating the cycle. In this way, the 5 ′ upstream sequence of the poly (A) sequence can be analyzed using the pyrosequencing method.

  It is also possible to prepare a mixed substrate solution lacking at least one base in advance and place it in the apparatus. In this case, it is not necessary to mix the substrate base.

  In this example, a system in which a limited extension reaction lacking one kind of base is performed twice in succession will be described.

  A glass substrate is used as the solid phase, and the template nucleic acid, primer DNA (oligo (dT) primer), washing buffer and the like to be measured are the same as those in Example 2. The oligo (dT) primer was immobilized on the slide glass substrate, the primer and template nucleic acid were hybridized, the washing conditions, and the restriction extension reaction conditions were the same as in Example 2 (FIG. 5 (a)). Before the base sequence measurement, as the extension reaction, the first stage is the combination of dTTP, dCTP, and dGTP (restriction extension reagent set 1). The second stage is the combination of dATP, dTTP, and dCTP. Perform an extension reaction using (Restricted Extension Reaction Reagent Set 2). FIG. 9 illustrates the extended state at each stage. FIG. 9 (a) shows the state after rehydration, FIG. 9 (b) shows the state after the first-stage restriction extension reaction, and FIG. 9 (c) shows the state after the second-stage restriction extension reaction. In the first stage, the remaining poly (A) region is extended, and further, 7 bases are extended from that point. The reaction of the second stage is further extended by 5 bases, and the base sequence is analyzed from a position 12 bases away from the end point of the poly (A) region. As sequence analysis means, detect by sequentially reacting with Cy3-dATP, Cy3-dCTP, Cy3-dGTP, Cy3-dTTP, Cy3-dATP, Cy3-dCTP, Cy3-dGTP, Cy3-dTTP, ... Thus, the base sequence can be determined continuously. In this case, each time the type of the base is changed, it is necessary to clean the substrate, bleach the phosphor, or desorb the phosphor molecule part. Moreover, what is labeled with a different phosphor for each base type may be used, and four types of phosphor-labeled bases may be mixed and reacted, and the fluorescence measurement may be detected by separating the four colors. In this case, in one reaction, it can be determined which base type is one base of the target nucleic acid, and rapid sequence analysis becomes possible.

  This example can also be applied to mRNA having a poly (A) region having the same structure, and a base sequence from about 12 bases away from the end point of the poly (A) region can be obtained efficiently. In the sequence immediately after the end point of the poly (A) region, even if the system is difficult to identify mRNA, it may be identified by changing the reading position. This example is effective in such a case. The distance from the end point of the poly (A) region varies depending on the type, number and order of the above-described restriction extension reactions, and the sequence of the target nucleic acid, but can be arbitrarily selected. These methods are effective, for example, as a method of immobilizing oligo (dT) primers, comprehensively hybridizing intracellular mRNA, and effectively detecting the sequence excluding the poly (A) region. In addition, it is effective to identify which mRNA is from the detected sequence using homology search and to obtain their frequency distribution, that is, mRNA expression distribution.

  Moreover, the above restriction | limiting extension reaction system is applicable also to the arrangement | sequence analysis of a general nucleic acid. For example, if there is a repetitive sequence of 2 bases that does not require sequence analysis after the hybrid position with the designed primer, it is effective to perform a restriction extension reaction method combining these 2 bases complementary before sequence analysis. An array can be obtained effectively.

  The present invention is useful for single molecule sequencing of mRNA and cDNA in genome analysis. Therefore, it can be used in all life sciences including biology, chemistry, medicine, and the like.

2 is an explanatory diagram of Embodiment 1. FIG. It is a figure which shows the base sequence analyzer in Example 4. FIG. It is explanatory drawing which shows the fluorescence intensity for every expansion | extension state in Example 1. FIG. It is explanatory drawing of the expansion | extension state in Example 1. FIG. 6 is an explanatory diagram of a state for each reaction process in Example 2. FIG. It is a block diagram of the fluorescence detection apparatus in Example 2. 6 is a schematic explanatory diagram of irradiation characters in a fluorescence detection state in Example 2. FIG. 3 is an example of a captured image of a high-sensitivity two-dimensional CCD camera obtained in Example 2. FIG. FIG. 6 is an extension state diagram at each reaction stage in Example 5.

Explanation of symbols

1a Oligo (dT) primer hybridized in the middle of the poly (A) region 1b 1a State in which dTTP and DNA polymerase reacted with 1a and the end of the oligo (dT) primer reached the 5 'end of the poly (A) region 1c 1b Sequence analysis using extension reaction 2a Poly (A) region 5 'end oligo (dT) primer hybridized 2b 2a Even if dTTP and DNA polymerase are added to oligo (dT) No extension from primer 2C 2b is used for sequence analysis using extension reaction 3 Reagent storage unit 3a dATP solution container 3b dCTP solution container 3c dGTP solution container 3d dTTP solution container 3e DNA polymerase reaction solution container 3f DNA polymerase and luminescence reaction solution container 3g Washing Buffer container 4 Dispensing unit 5 Liquid feeding tube 6 Reagent inlet 7 Heating / cooling device 8 Reaction substrate 9 Flow chamber 10 Waste liquid tube 11 Waste liquid container 12 Two-dimensional sensor camera (high sensitivity cooling CCD camera)
13 Two-dimensional sensor camera controller 14 Control PC
15 Monitor

SEQ ID NO: 1-description of artificial sequence: template nucleic acid SEQ ID NO: 2-description of artificial sequence: oligo dT primer

Claims (13)

A nucleotide sequence analysis method comprising the following steps:
1) Hybridize the primer to the nucleic acid to be analyzed;
2) A reaction reagent lacking at least one base of dATP, dCTP, dGTP, and dTTP is added to the nucleic acid to be analyzed, and an extension reaction is performed with DNA polymerase;
3) After the extension reaction, the reaction reagent is washed;
4) A sequence analysis reaction using a nucleic acid extension reaction is performed to determine a desired region away from the primer binding site on the nucleic acid to be analyzed.   The method according to claim 1, wherein the steps 2) and 3) are performed a plurality of times while changing the type of the defective base.   The method according to claim 1, wherein the nucleic acid to be analyzed is mRNA having a poly (A) region or cDNA derived from mRNA, and the primer is an oligo (dT) primer.   The analysis target is mRNA having a poly (A) region, the primer is an oligo (dT) primer, and an extension reaction is performed in a reaction system lacking at least one base other than dTTP, The method of claim 1.   The analysis target is cDNA derived from mRNA and having a poly (T) region, the primer is an oligo (dA) primer, and an extension reaction is performed in a reaction system lacking at least one base other than dATP. The method of claim 1, wherein:   The method according to claim 4, wherein the extension reaction is performed a plurality of times while changing the type of the deficient base.   6. The method according to claim 5, wherein the extension reaction is performed a plurality of times while changing the type of the deficient base.   The method according to claim 1, wherein a desired region is sequenced without amplification of the nucleic acid to be analyzed. A nucleotide sequence analysis method comprising the following steps:
1) Hybridizing oligo (dA) primer to cDNA derived from mRNA and having a poly (T) region;
2) An extension reaction is performed by adding DNA polymerase and dATP to the cDNA;
3) After the extension reaction, DNA polymerase and dATP are removed from the reaction system;
4) Sequencing is performed by conducting a sequence analysis reaction utilizing a nucleic acid extension reaction. A nucleotide sequence analysis method comprising the following steps:
1) Hybridize oligo (dT) primer to mRNA with poly (A) region;
2) An RNA-dependent DNA polymerase and dTTP are added to the mRNA to perform an extension reaction;
3) After the extension reaction, RNA-dependent DNA polymerase and dTTP are removed from the reaction system;
4) Sequencing is performed by conducting a sequence analysis reaction utilizing a nucleic acid extension reaction. A base sequence analyzer for sequencing a desired region away from a primer binding site on a nucleic acid to be analyzed:
A chamber for performing an extension reaction from the primer of the nucleic acid to be analyzed and a sequence analysis reaction;
A container and storage unit for storing the four substrate bases and reagents necessary for the reaction;
A reagent supply system for supplying the substrate base and a reagent necessary for the analysis to the chamber in any combination in a state lacking at least one base;
A heating and cooling system for controlling the temperature in the chamber;
A detection system for detecting the sequence analysis reaction;
A base sequence analyzing apparatus comprising: an analysis system for analyzing the detection result.   Furthermore, the base sequence analyzer of Claim 11 containing the container for storing 4 types of label | marker dNTPs. A base sequence analyzer for sequencing a desired region away from a primer binding site on a nucleic acid to be analyzed:
A chamber for performing an extension reaction from the primer of the nucleic acid to be analyzed and a sequence analysis reaction;
A container and storage unit for storing one or more substrate base mixed reagents lacking at least one base, reagents necessary for the reaction, and four dNTPs or four labeled dNTPs, respectively;
A reagent supply system for supplying the reagent in the storage unit to the chamber;
A heating and cooling system for controlling the temperature in the chamber;
A detection system for detecting the sequence analysis reaction;
A base sequence analyzing apparatus comprising: an analysis system for analyzing the detection result.

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