Method for analyzing base sequence of nucleic acid

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Technical field to which the invention belongs]

The present invention relates to a method for identifying a nucleobase sequence using a DNA chip used for DNA diagnosis and treatment.

【0002】

[Previous technology]

One of the methods for determining the sequence of a substance such as nucleic acid or checking the sequence is to use a DNA array. USP5445934 discloses a DNA array in which more than 100,000 oligonucleotide probes are bound to an inch square. Such a DNA array has an advantage that many items can be tested at one time with a small amount of sample. When a fluorescently labeled sample is passed on such a DNA chip, a DNA fragment having a sequence complementary to the probe on the DNA chip binds to the probe, and only that portion can be identified by fluorescence, and the DNA sample can be identified. The sequence of DNA fragments can be elucidated.

【0003】

The Sequencing by Hybridization (SBH) method is a method for examining a base sequence using such a DNA array, and the details thereof are described in USP5202231. In the SBH method, the entire sequence of oligonucleotides possible for a certain length is arranged on a substrate to detect a completely complementary hybrid formed by a hybridization reaction with a sample DNA, which is completely complementary. Once a set of hybrids is obtained, the set should be a set of sequences deviated by 1 base for a certain sequence, and the determination is made by analyzing them.

【0004】

In principle, in order to investigate whether or not a specific sequence is present in the sample DNA, a hybridization reaction is carried out using a sequence complementary to that sequence as a probe to examine the presence or absence of binding. However, in reality, it is very difficult to check the presence or absence of a hybrid with one type of probe and judge it as one inspection item based on it. This is because, when comparing completely complementary hybrids, the fluorescence derived from the hybrids has different intensities depending on each sequence. In particular, the GC content in the base sequence has a great influence on the stability of the hybrid. Moreover, even sequences that are not completely complementary and contain a single base mismatch form a hybrid and fluoresce. It is a common phenomenon that the hybrids are less stable and less fluorescent than the perfect match when compared between the same sequences, but have higher fluorescence intensity than other fully complementary hybrids. is there. Moreover, even if it is a single nucleotide mismatch, its stability changes greatly depending on the position of the hybrid containing the mismatch. When a mismatch is included at the end, a relatively stable hybrid is obtained, but when it is contained in the middle of the hybrid, the continuous portion of the complementary strand is divided, which makes it unstable. As described above, various factors are intertwined with respect to the stability of the hybrid body, and the absolute value (reference value) of the fluorescence intensity for determining whether or not the hybrid is completely complementary cannot be obtained at present. In addition, it can be said that the condition that the single base mismatch is completely eliminated and the fluorescence can be detected only from the perfect match has not been obtained.

【0005】

Proc. Natl. Acad. Sci. USA Vol.82, pp1585-1588 (1985) describes a method using tetramethylammonium chloride as a device for eliminating the difference in the stability of the hybrid depending on the sequence. However, the above problems have not been completely solved.

【0006】

Therefore, as a method of determining whether or not it is a perfect match, in Science vol.274 p610-614, 1996, a sequence in which a 1-base mismatch is arranged in the middle of the probe sequence of a 15-mer oligonucleotide is prepared, and a perfect match and a 1-base are prepared. A method of comparing the fluorescence intensity derived from a hybrid with a mismatch and determining that the perfect match is positive when the intensity is stronger is described.

【0007】

Further, USP5733729 discloses a method for knowing the base sequence of a sample from a comparison of the fluorescence intensities of the obtained hybrids using a computer as a method for making a more accurate determination in addition to the above method. There is.

【0008】

In these methods, a nucleic acid base portion of the location to be examined is set in the middle of the probe, always four nucleotides set to its position providing a preparative, and for such probes set 1 nucleotide each shifted sequences It is necessary to prepare. Then, using the 15mer oligonucleotide as described above, the stability of each is theoretically determined by comparing with other three types of probes that have a 1-base mismatch in the middle to determine whether or not they are perfect matches. It is said that more accuracy can be obtained by evaluating it either empirically or empirically. If the length of the base in the region to be examined is L, the number of probes is 4 × L (20 types if 5 bases).

【0009】

[Problems to be Solved by the Invention]

The method using the above mismatch is relatively easy to determine by comparing with a 1-base mismatch at the same position in the same sequence, and the number of probes may be small (in SBH, 1024 types are used for similar analysis). Although it is an excellent method in terms of (requires a probe), it has a serious drawback that accurate information cannot be obtained when there is a two-base mismatch in the same region, or when there is a base defect or insertion.

【0010】

On the other hand, the SBH method solves the above problems and can deal with any mutation in principle, but its determination is quite difficult. The stability of the hybrid depends on the strength of the 1-base mismatch of another sequence rather than the perfect match of one sequence, and the position of the mismatch in the sequence even though it is a 1-base mismatch. Due to the large difference in sex. As a result, perfect match, 1-base mismatch, and 2-base mismatch (continuous, discontinuous) cannot be simply judged from the fluorescence intensity, such as theoretical prediction, comparison with various sequences, and accumulation of empirical parameters. Requires complex analysis.

【0011】

Furthermore, in order to measure the strength of the hybrid of each probe and analyze the value to determine and determine the gene sequence, a large-scale computer device is required in addition to the detection device that reads the array, and the DNA array It is a big obstacle to perform a simple genetic diagnosis using.

【0012】

In view of these problems, the present invention provides a method for accurately sequencing a gene without requiring complicated analysis.

【0013】

[Means for solving problems]

As mentioned above, the intensity of the hybrid is governed by various factors, and when a probe with a length of about 15 mer to 20 mer is used, it is not possible to completely eliminate the fluorescence intensity of the hybrid having a 1-base mismatch. It's virtually difficult. On the other hand, for a sequence having a 2-base mismatch, it is relatively easy to obtain conditions for suppressing the formation of a hybrid regardless of the position, continuity, or discontinuity of the 2-base mismatch.

【0014】

The present invention has been made based on such a finding, and in addition to the spots of a perfect match sequence, the spots of a hybrid having a predetermined number of mismatches, for example, a sequence of a single nucleotide mismatch are also regarded as positive. Has one feature.

【0015】

That is, the method for identifying an unknown base sequence at a predetermined site of the target single-stranded nucleic acid according to one embodiment of the present invention is (a) Each of the multiple types of single-stranded nucleic acid probes Nos. 1 to n (n 2) having a base sequence complementary to each of the expected multiple base sequences of the unknown base sequence The process of preparing probe arrays arranged on a substrate so as to be isolated from each other; (b) With a first sample containing a labeled single-stranded nucleic acid having a base sequence completely complementary to the base sequence of one of the single-stranded nucleic acid probes and being fluorescently labeled. The probe array is reacted under the condition that the single-stranded nucleic acids complementary to each other form a double-stranded nucleic acid to remove the unreacted labeled single-stranded nucleic acid, and then the fluorescence observed from the array is observed. The step of measuring the intensity for each single-stranded nucleic acid probe on the probe array to obtain a first template pattern showing the relationship between the position of each single-stranded nucleic acid probe on the probe array and its fluorescence characteristics; (c) For each of the other single-stranded nucleic acid probes, perform the same operation as in step (b), and the single-stranded nucleic acid probe on the probe array is completely complementary to each base sequence. The step of obtaining the second to nth template patterns showing the relationship between the position of each single-stranded nucleic acid probe on the probe array when the single-stranded nucleic acid and the double-stranded nucleic acid are formed and their fluorescence characteristics; (d) The probe array and the second sample containing the target single-stranded nucleic acid are reacted under the same conditions as those in which the template pattern was obtained, and then the presence / absence and intensity of fluorescence are determined respectively on the probe array. The step of measuring the single-stranded nucleic acid probe of the base sequence of the above and obtaining a sample pattern showing the relationship between the position of each single-stranded nucleic acid probe on the probe array and its fluorescence characteristics; (e) The sample pattern is compared with the n template patterns obtained in the above steps (b) and (c), and when there is a template pattern that substantially matches the pattern, it is used to create the template pattern. It is characterized by having a step of identifying the base sequence of the single-stranded nucleic acid as an unknown base sequence of the target single-stranded nucleic acid.

【0016】

In addition, another embodiment of the present invention has one feature in that, for example, a threshold is provided between the fluorescence intensity of the 1-base mismatch and the fluorescence intensity of the 2-base mismatch hybrid to distinguish between positive and negative. Another such embodiment is a method of identifying an unknown base sequence at a predetermined site of a target single-stranded nucleic acid. (a) Each of the multiple types of single-stranded nucleic acid probes Nos. 1 to n (n 2) having a base sequence complementary to each of the expected multiple base sequences of the unknown base sequence The process of preparing probe arrays arranged on a substrate so as to be isolated from each other; (b) A first sample containing a labeled single-stranded nucleic acid having a base sequence completely complementary to the first base sequence of the single-stranded nucleic acid probe and the probe array. Are reacted under the condition that the single-stranded nucleic acids complementary to each other form a double-stranded nucleic acid, and the observed fluorescence is measured for the single-stranded nucleic acid probe of each base sequence on the probe array. A step of obtaining a template pattern I showing the relationship between the position of each single-stranded nucleic acid probe on the probe array and its fluorescence characteristics; (c) The obtained first template pattern is analyzed, and the average value (F) of the fluorescence amount of the double-stranded nucleic acid having the mismatch base pair number (i) with the probe at each position is analyzed._i) Is calculated; (d) Fluorescence amount of fully complementary double-stranded nucleic acid without mismatch (F)₀) And the average value of the fluorescence amount of the double-stranded nucleic acid having a 1-base mismatch (F).₁) And the difference (F)_1,0), And the fluorescence amount (F) of the double-stranded nucleic acid having (i + 1) base mismatch._{i + 1}) And i-base mismatch fluorescence amount (F)_i) Difference (F_{i + 1, i}), F_{i, i + 1} <<F_{i-1, i}The process of setting i so that (e) With respect to the base sequence of the second probe, the position on the substrate of the single-stranded nucleic acid probe having a base sequence in which the number of mismatched base pairs is i or less is made positive, and a mismatch of i + 1 or more is made. The step of obtaining a template pattern II in which the position of the probe having a base sequence on the substrate is negative and the positive position is formed; (f) The same operation as in (e) is performed on all other single-stranded nucleic acid probes, and the position on the substrate of the single-stranded nucleic acid probe having a base sequence in which the number of mismatched base pairs is i or less is formed. Step to obtain template patterns III ~ n; (g) The probe array and the sample containing the target single-stranded nucleic acid are reacted under the same conditions as the conditions for obtaining the first template pattern, and the presence / absence and intensity of fluorescence are adjusted for each of the probe arrays. A step of measuring a single-stranded nucleic acid probe of a base sequence and obtaining a pattern showing the relationship between the position of each single-stranded nucleic acid probe on the probe array and its fluorescence characteristics; (h) Compare the pattern with the n template patterns obtained in the above steps (b) (e) and (f), and if there is a template pattern that substantially matches the pattern, correspond to the template pattern. It is characterized by having a step of specifying the base sequence of the single-stranded nucleic acid to be used as an unknown base sequence of the target single-stranded nucleic acid.

【0017】

Then, by adopting such an embodiment, a pattern formed by the spots distinguished from the positive on the substrate can be obtained as an image, and the sequence can be analyzed by comparing it with the expected pattern, which is unknown. The gene sequence can be easily identified.

【0018】

Further, in the present invention, a hybridization reaction condition for completely distinguishing such a 1-base mismatch and a 2-base mismatch will be presented.

【0019】

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described. (Determination by Fluorescent Image) In one embodiment of the present invention, it is particularly effective when nucleobases that may cause a mismatch are present in close proximity. Here, the base sequence corresponding to the 238th and 239th amino acid sequences of the tumor suppressor gene p53 is included.^5'GATGGGNCTCNNGTTCAT^3'Will be described as an example. The above example is one form for explaining the outline of the invention, and presents the same concept in that the present invention processes an array in any form as an image. It does not limit the arrangement of. As a matter of course, the SBH method is also the subject of analysis of the present invention.

【0020】

In the above example, when a complete set of probes in which each base part marked with N is replaced with four types of bases (A, G, C, T) is prepared, that is, three locations (not necessarily continuous). To find out about the bases of 4³= 64 types, 4 in the case of 5 places⁵= 1024 types of probes are lined up on the board.

【0021】

Figure 1 shows an example of placement when 64 types of probes are used.

【0022】

A probe (probe number 1 to 16) whose first N is A is placed in the upper left region of the 64 probe array divided into four, and a probe (probe number 17 to 32) whose first N is G is placed in the lower left. Be placed. Similarly, the upper right is C (probe number 33 to 48), and the lower right is T (probe number 49 to 64). Within each region, the probe with the second N of A is located in the first row, G is in the second row, C is in the third row, and T is in the fourth row. Also, the probe whose third N is A is placed in the first row counting from the top, the sequence where G is placed is placed in the second row, and C is placed in the third row and T is placed in the fourth row. .. As a result, for example^5'GATGGGACTCAAGTTCAT^3'Such an array corresponds to the upper left spot. In addition, it is a sequence corresponding to a normal gene.^5'ATGAACCGGAGGCCCATC^3'Is the probe DNA located in the third column from the right and the third row from the top.^5'GATGGGCCTCCGGTTCAT^3'It is expected to form a hybrid with.

【0023】

The case where the single base mismatch is treated as a positive spot will be described below. In this case, assuming that the perfect match sequence is probe No. 42 (normal gene), the 1-base mismatch sequences judged to be positive are 9 filled places, as shown in Fig. 2 together with the perfect match. It is expected to form a pattern.

【0024】

On the other hand, in the mutant sequence for the sequence to be specified of the target nucleic acid, for example, the pattern change as shown in FIG. 3 is observed.

【0025】

In the present invention, an expected fluorescence pattern image consisting of such a perfect match and a single nucleotide mismatch is input to a storage device such as a computer in advance, and a determination is made by comparison with a fluorescence image obtained by a predetermined method. At this time, detailed quantitative data of the fluorescence intensity of each positive spot is not required. Only positive and negative judgments for a certain threshold value are sufficient, and simple and automated judgments using a computer or the like are possible.

【0026】

(Setting of threshold value) When using a probe of about 18 mer, it is preferable to set the threshold value between the fluorescence intensity due to a 1-base mismatch and the fluorescence intensity due to a 2-base mismatch. The fluorescence intensity varies depending on the sequence composition and reaction conditions, but the highest fluorescence intensity (usually a perfect match) is set at a value of 50% to 25%, more preferably 30% to 20% as a threshold value. Then it is good. If the probe length is short, the threshold will be even lower.

【0027】

Those containing a 3-base mismatch have a fluorescence amount of 10% or less of the maximum value and can be completely distinguished.

【0028】

When the threshold value is set to 1/4 of the maximum fluorescence amount, the distribution of fluorescence intensity is predicted by setting the complete match sequence and the 1-base mismatch sequence to 4, the 2-base mismatch sequence to 1 and the 3-base mismatch sequence to 0. become that way.

【0029】

The above example will be described with respect to a more specific determination method.

【0030】

If the hybridization reaction proceeds very selectively, the strong fluorescence will be concentrated at one point (perfect match). Next, when the sensitivity is gradually increased, as expected from FIG. 3 in the above arrangement example, the 1-base mismatch should be arranged in vertical and horizontal lines centered on the perfect match. However, the actual fluorescence image is not always arranged in a line with three vertical and horizontal lines centered on a strong spot. Due to the difference in stability between single base mismatches, not all 6 have the same intensity, so some spots may not be detected, but at least some spots should be visible in these lines. At that time, the remaining three 1-base mismatches can be detected even though there are intensity shades at the expected positions.

【0031】

Also, sometimes, a perfect match and a 1-base mismatch give the same fluorescence intensity, and from the beginning, an image close to the expected image consisting of 10 spots of the perfect match and the 1-base mismatch can be obtained.

【0032】

The two-base mismatch sometimes exceeds the threshold value, but even in such a case, it can be easily discriminated as a deviation from the expected pattern.

【0033】

In this way, the method of the present invention, which determines by comparing the predicted pattern with the actually obtained fluorescence image, can easily determine the presence or absence of mutation in the sample gene, and any base (s) are mutated to what. It has the feature that it is possible to determine the content of the mutation at the same time.

【0034】

In addition, the idea of evaluating the results of hybridization reactions using 64 types of probes by pattern is advantageous in terms of more reliable judgment than the case of judging by only one spot. Hybrids with 64 DNA probes have different thermal stability depending on their sequences, so there is no guarantee that a perfect match will be overwhelmingly stable and emit strong fluorescence. In addition, it is often not possible to determine which is the strongest spot and the perfect match spot due to dust on the substrate or artifacts during the hybridization reaction. The judgment based on the point pattern can compensate for some variation in the amount of fluorescence.

【0035】

(Probe length) The probe length used in the present invention is about 8 mer to 30 mer, more preferably 12 mer to 25 mer. Below 8 mer, the stability of hybrids with a single nucleotide mismatch is low, and the amount of fluorescence derived from the perfect match is predominant. Also, for probes longer than 30 mer, the fluorescence of the 2-base mismatch is stronger than the 1-base mismatch in some cases (for example, when there are mismatch sites at both ends).

【0036】

(Hybridization reaction conditions) As the conditions for the hybridization reaction that gives the above-mentioned good threshold value, the entire substrate is heated while being immersed in the sample solution, and both the DNA probe and the sample DNA on the substrate are simultaneously heat-denatured. After that, it is gradually cooled and the hybridization reaction is carried out at a slightly higher temperature. The salt concentration during the reaction is preferably 100 mM or less.

【0037】

The temperature for heat denaturation is 60 ° C or higher, preferably 80 ° C or higher. The temperature setting for heat denaturation depends on the stability of the DNA array substrate itself, the length and concentration of the sample, and the type of labeled compound. For example, in a substrate to which a resin is applied and the resin is reacted with DNA to be bonded, the resin layer may be destroyed by raising the temperature. On the other hand, the substrate in which the silane coupling agent is used in the manufacturing process is relatively stable to heat and can be heated to a higher temperature. When the sample DNA is single-stranded, the intramolecular double-strand structure is considered to be almost eliminated at 70 ° C or higher, but when the sample DNA is double-stranded or the sample DNA is long single-stranded DNA, it is considered that the double-strand structure is almost eliminated. It is necessary to further raise the temperature or add a denaturing agent such as formamide to promote the dissociation into a single chain. The time required for heat denaturation is 10 minutes or more, preferably about 30 minutes.

【0038】

The conditions for the hybridization reaction are carried out by considering the probe length, sequence, and type of sample, and changing the temperature and salt concentration according to a conventional method. Preferably used as a condition for distinguishing and recognizing extremely similar sequences as in the present invention is 3 hours or more at 45 ° C. in a solution containing 100 mM salt. However, the reaction time is greatly affected by the sample concentration and is not limited to the above reaction conditions. If the sample has a high concentration, it can be sufficiently determined within 3 hours, and if the sample solution is dilute, a reaction time of 10 hours or more is required. When adding formamide, it is necessary to increase the salt concentration. (Production method of DNA array substrate) An example of a method for producing a DNA array capable of satisfactorily advancing this hybridization reaction is shown below. However, the purpose of the present invention is to show a simple method for evaluating the hybridization pattern on the substrate and determining the base sequence of the sample, and basically, the method for producing the substrate is not particular.

【0039】

In a DNA array, the DNA probe is covalently fixed by reaction with a functional group on the surface of the substrate. As a mode of binding between the functional group and DNA, for example, a method of causing a binding reaction between a maleimide group on the glass surface and an SH group at the end of DNA will be described.

【0040】

As a method for introducing a maleimide group, first, an aminosilane coupling agent is reacted with a glass substrate, and then the maleimide group is introduced by a reaction between the amino group and an EMCS reagent (N- (6-Maleimidocaproyloxy) succinimide: manufactured by Dojin). Introduce. The introduction of SH groups into DNA can be performed by using a 5'-Thiol-Modifier C6 (manufactured by Glen Research) on an automatic DNA synthesizer.

【0041】

A spot is formed on the substrate by the inkjet method of the DNA solution, and the probe DNA is immobilized by the reaction between the maleimide group on the DNA substrate and the SH group at the end of the DNA.

【0042】

As a DNA solution suitable for ejection to a glass substrate having a maleimide group by an inkjet method, a solution containing glycerin, urea, thiodiglycol or ethylene glycol, acetylenol EH (manufactured by Kawamura Fine Chemicals Co., Ltd.), and isopropyl alcohol is preferable. In particular, a solution containing 7.5% glycerin, 7.5% urea, 7.5% thiodiglycol, and 1% acetylenol EH (all by mass) is preferable.

【0043】

The array substrate to which DNA is bound is immersed in a 2% bovine serum albumin aqueous solution for 2 hours to perform a blocking reaction, and then used for a hybridization reaction.

【0044】

[Example]

The following will be described in more detail with reference to Examples. (Example 1: Pattern recognition I) 1. Probe design Among the mutations in the nucleotide sequence CGGAGG corresponding to the 248th and 249th amino acid sequences of the p53 gene, which is a tumor suppressor gene, the first C is T and the second A is G. And it is known that the third G of the 249th amino acid is mutated to T. Therefore, we designed 64 types of probes focusing on these three base sequences.

【0045】

In other words, the total length of the probe is 18 mer, 6 bases containing this mutation are located in the middle, and the front and back are sandwiched by a common sequence.^5'ATGAACNNGAGNCCCATC^3'Is. Here, the part represented by N corresponds to four kinds of nucleobases, A, G, C, and T. Since the probe DNA is a sequence complementary to the sequence to be detected (the above sequence), in practice,^5'GATGGGNCTCNNGTTCAT^3'Will be.

【0046】

Figure 1 shows the layout of 64 DNA probes on the DNA array. Each sequence (SEQ ID NO: 1 to 64) is specifically shown in Table 1.

【0047】

[table 1]

<img file="JP2002306166A_D0001.tif" />It is a sequence corresponding to a normal gene^5'ATGAACCGGAGGCCCATC^3'Is the 42nd probe DNA located in the 3rd column from the right and the 3rd row from the top.^5'GATGGGCCTCCGGTTCAT^3'It is expected to form a hybrid with.

【0048】

In 64 experiments, fluorescence from hybrids with 1-base mismatch is expected in addition to perfect match. Figure 2 shows the expected fluorescence pattern consisting of a perfect match and a single base mismatch.

【0049】

2. Preparation of maleimide group-introduced substrate (Substrate cleaning) A 1-inch square glass plate was placed in a rack and immersed in an ultrasonic cleaning detergent overnight. Then, ultrasonic cleaning was performed in the detergent for 20 minutes, and then the detergent was removed by washing with water. After rinsing with distilled water, sonication was further performed for 20 minutes in a container containing distilled water. Next, it was immersed in a preheated 1N sodium hydroxide solution for 10 minutes. Subsequently, water washing and distilled water washing were performed.

【0050】

(Surface treatment) A 1% silane coupling agent aqueous solution (manufactured by Shin-Etsu Chemical Co., Ltd., trade name KBM603) was immersed at room temperature for 20 minutes, and then nitrogen gas was sprayed on both sides to remove water and dry. The silane coupling process was completed by baking in an oven heated to 120 ° C for 1 hour. Subsequently, 2.7 mg of EMCS (N- (6-Maleimidocaproyloxy) succinimide: manufactured by Dojin) was weighed and dissolved in a 1: 1 solution of DMSO / ethanol (final concentration 0.3 mg / ml). The glass substrate treated with the silane coupling agent was immersed in this EMCS solution for 2 hours to react the amino group of the silane coupling agent with the carboxyl group of the EMCS solution. In this state, a maleimide group derived from EMCS is present on the glass surface. The glass plate reacted with the EMCS solution is washed with distilled water, dried with nitrogen gas, and used for the binding reaction with DNA. 3. DNA binding reaction to the substrate (Synthesis of 64 types of DNA probes) The above 64 types of probe DNAs having an SH group (thiol group) at the 5'end were synthesized by requesting Bex Co., Ltd. (Discharge of DNA probe) The following discharge operation was performed using each of the above 64 types of DNA. Each DNA was dissolved in water and diluted with SG clear (an aqueous solution containing 7.5% glycerin, 7.5% urea, 7.5% thiodiglycol, and 1% acetylenol EH) to a final concentration of 8 μM. The nozzle of the BJ printer head BC62 (manufactured by Canon Inc.) modified so that a small amount of sample could be ejected was filled with 100 μl of this DNA solution. Using 2 heads so that 6 types of DNA can be ejected for each head, 12 types of DNA are ejected at a time, and the heads are exchanged 6 times, and each spot of 64 types of DNA is formed independently. It was discharged so as to be.

【0051】

Each probe was set so that the spot diameter was 70 μm and the pitch was 200 μm, and 64 types were spotted in an 8 × 8 matrix. Then, it was left in a humidifying chamber for 30 minutes, and a reaction was carried out to bind the probe DNA to the substrate. Hybridization reaction (Blocking reaction) After completion of the reaction, the substrate was washed with a 1MNaCl / 50 mM phosphate buffer (pH 7.0) solution, and the DNA solution on the glass surface was completely washed away. Then, it was immersed in 2% bovine serum albumin aqueous solution and left for 2 hours to carry out a blocking reaction. (Synthesis of model sample DNA) Labeled DNA No. 1 having the normal sequence of the p53 gene and having the same region and the same length as the probe DNA was prepared. The sequence is as shown below, with rhodamine bound to the 5'end.

【0052】

No.1:^5'Rho-ATGAACCGGAGGCCCATC^3'(Hybridization reaction conditions) 2 ml of a 10 nM model sample DNA solution containing 100 mM NaCl is placed in a bag for a hybridization reaction containing a DNA array substrate, first heated at 80 ° C for 10 minutes, and then the temperature of the incubator is 45 ° C. And let it react for 15 hours as it was. 5. Detection (Method) Detection was performed by connecting an image analysis processing device ARGUS (manufactured by Hamamatsu Photonics) to a fluorescence microscope (manufactured by Nikon Corporation). (Results) Fig. 4 shows the amount of fluorescence obtained as a result of the hybridization reaction with the labeled DNA No. 1 of 18mer, which is a model system. The maximum amount of fluorescence is probe 42, which is completely complementary. The amount of fluorescence is set to the maximum value (100%), a threshold is set at 20%, and the areas above that are painted black.

【0053】

It can be seen that the spots in No10, 26, and 58 also have fluorescence, which is in good agreement with the expected pattern Fig. 2. By further lowering the threshold, it matches the expected pattern. In other words, in addition to the above three spots, the part of the 1-base mismatch sequence is lined up in vertical and horizontal lines centering on the perfect match.

【0054】

(Example 2: Pattern recognition II) A DNA array substrate consisting of 64 types of probes was prepared in the same manner as in Example, and a hybridization reaction was performed with a rhodamine-labeled DNA having the No. 2 sequence as a model sample. The sequence of No. 2 is complementary to the No. 46 probe of FIG.

【0055】

No.2:^5'Rho-ATGAACCAGAGGCCCATC^3'The hybridization reaction conditions are the same as in Example 1.

【0056】

Similar to Example 1, the expected pattern was obtained in Fig. 5. On the other hand, the obtained results are shown in Fig. 6. The threshold value is set to 10% of the maximum value, and the result of the hybridization reaction is shown in black. Good correspondence with expectations.

【0057】

(Example 3: Pattern recognition III) The same experiment as in Example 2 was performed using the same model sample DNA as in Example 2. However, the sample DNA concentration used for the hybridization reaction was set to 5 nM, and the reaction was carried out overnight at 40 ° C. The results obtained are shown in Fig. 7.

【0058】

When the threshold is set to 50%, fluorescence appears at the positions of probes 34 and 62 (shaded areas) of the 1-base mismatch, and when the threshold is further lowered to 30%, the result matches the expected pattern. In this case, a 2-base mismatch of NO.6 and 22 is also detected, but as a deviation from the pattern composed of the 1-base mismatch, it can be determined that it is a 2-base mismatch, and the spot of the perfect match is No. 46. Can be determined.

【0059】

[Effect of the invention]

As described above, as compared with the conventional method of determining only by the presence or absence of the hybrid, the method of the present invention further enables more accurate detection by taking into consideration the fluorescence amount of the single nucleotide mismatch.

【0060】

Since the hybrid with the DNA probe has different thermal stability depending on each sequence, there is no guarantee that the perfect match is overwhelmingly stable and emits strong fluorescence. Judgment by pattern is advantageous in terms of more reliable judgment than the case of judgment by only one spot.

【0061】

Due to dust on the substrate and artifacts during hybridization reactions, it is often not possible to determine which is the strongest spot and the perfect match spot. In that respect, the determination based on the pattern in the present invention can compensate for a slight variation in the amount of fluorescence.

【0062】

Therefore, according to the present invention, it is possible to provide a test method capable of screening for gene mutations easily and efficiently.

【0063】

[Sequence list]

SEQUENCE LISTING &lt;110&gt; Canon INC. &lt;120&gt; A method of analyzing a base sequence of a nucleic acid &lt;130&gt; 3888012 &lt;160&gt; 64 &lt;210&gt; 1 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 1 gatgggactc aagtt cat &lt;210&gt; 2 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 2 gatgggactc aggtt cat &lt;210&gt; 3 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 3 gatgggactc acgtt cat &lt;210&gt; 4 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 4 gatgggactc atgtt cat &lt;210&gt; 5 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 5 gatgggactc gagtt cat &lt;210&gt; 6 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 6 gatgg gactc gggtt cat &lt;210&gt; 7 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 7 gatgggactc gcgttcat &lt;210&gt; 8 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 8 gatgggactc gtgttcat &lt;210&gt; 9 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 9 gatgggactc cagttcat &lt;210&gt; 10 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 10 gatgggactc cggttcat &lt;210&gt; 11 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 11 gatgggactc ccgttcat &lt;210&gt; 12 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 12 gatgggactc ctgttcat &lt;210&gt; 13 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 13 gatgggactc tagttcat &lt;210&gt; 14 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 14 gatgggactc tggttcat &lt;210&gt; 15 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 15 gatgggactc tcgttcat &lt;210&gt; 16 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 16 gatgggactct tgttcat &lt;210&gt; 17 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 17 gatggggctc aagttcat &lt;210&gt; 18 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 18 gatggggctc aggttcat &lt;210&gt; 19 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 19 gatggggctca cgttcat &lt;210&gt; 20 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 20 gatggggctc atgttcat &lt;210&gt; 21 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 21 gatggggctcg agttcat &lt;210&gt; 22 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 22 gatggggctc gggttcat &lt;210&gt; 23 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 23 gatggggctc gcgttcat &lt;210&gt; 24 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 24 gatggggctc gtgttcat &lt;210&gt; 25 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 25 gatggggctc cagttcat &lt;210&gt; 26 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 26 gatggggctc cggttcat &lt;210&gt; 27 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 27 gatggggctc ccgttcat &lt;210&gt; 28 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 28 gatggggctc ctgttcat &lt;210&gt; 29 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 29 gatggggctct agttcat &lt;210&gt; 30 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 30 gatggggctc tggttcat &lt;210&gt; 31 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 31 gatggggctc tcgttcat &lt;210&gt; 32 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 32 gatggggctc ttgttcat &lt;210&gt; 33 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 33 gatgggcctc aagttcat &lt;210&gt; 34 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 34 gatgggcctc aggttcat &lt;210&gt; 35 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 35 gatgggcctc acgttcat &lt;210&gt; 36 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 36 gatgggcctc atgttcat &lt;210&gt; 37 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 37 gatgggcctc gagttcat &lt;210&gt; 38 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 38 gatgggcctc gggttcat &lt;210&gt; 39 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 39 gatgggcctc gcgttcat &lt;210&gt; 40 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 40 gatgggcctc gtgttcat &lt;210&gt; 41 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 41 gatgggcctc cagttcat &lt;210&gt; 42 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 42 gatgggcctc cggttcat &lt;210&gt; 43 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 43 gatgggcctc ccgttcat &lt;210&gt; 44 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 44 gatgggcctc ctgttcat &lt;210&gt; 45 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 45 gatgggcctc tagttcat &lt;210&gt; 46 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 46 gatgggcctc tggttcat &lt;210&gt; 47 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 47 gatgggcctc tcgttcat &lt;210&gt; 48 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 48 gatgggcctc ttgttcat &lt;210&gt; 49 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 49 gatgggtctc aagttcat &lt;210&gt; 50 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 50 gatgggtctc aggttcat &lt;210&gt; 51 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 51 gatgggtctc acgttcat &lt;210&gt; 52 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 52 gatgggtctc atgttcat &lt;210&gt; 53 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 53 gatgggtctc gagttcat &lt;210&gt; 54 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 54 gatgggtctc gggttcat &lt;210&gt; 55 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 55 gatgggtctc gcgttcat &lt;210&gt; 56 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 56 gatgggtctc gtgttcat &lt;210&gt; 57 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 57 gatgggtctc cagttcat &lt;210&gt; 58 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 58 gatgggtctc cggttcat &lt;210&gt; 59 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 59 gatgggtctc ccgttcat &lt;210&gt; 60 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 60 gatgggtctc ctgttcat &lt;210&gt; 61 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 61 gatgggtctc tagttcat &lt;210&gt; 62 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 62 gatgggtctc tggttcat &lt;210&gt; 63 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 63 gatgggtctc tcgttcat &lt;210&gt; 64 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt; &lt;223&gt; Sample origonucleotide &lt;400&gt; 64 gatgggtctc ttgttcat

[Simple explanation of drawings]

[Figure 1]

An arrangement example when 64 types of probes are used is shown.

[Figure 2]

It is a figure which shows the arrangement pattern of the region determined to be positive on the substrate with respect to the sequence of a target nucleic acid.

[Fig. 3]

It is a figure which shows the arrangement pattern of the region determined to be positive on the substrate in the mutant sequence with respect to a target nucleic acid.

[Fig. 4]

It is a figure which shows the pattern of the amount of fluorescence obtained in Example 1.

[Fig. 5]

It is a figure which shows the pattern expected in Example 2.

[Fig. 6]

It is a figure which shows the pattern of the fluorescence amount obtained in Example 2 at a threshold value of 10%.

[Fig. 7]

It is a figure which shows the pattern of the amount of fluorescence obtained in Example 3.