JP2006109841A - Screening method for variant gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method suitable for mass screening for a rapid determination of only the presence or absence of variation of gene without needing an expensive device and a complicated analysis. <P>SOLUTION: The invention relates to the screening method for variant gene comprising at least following steps, a first step preparing an array on a solid phase arranged to comprises a completely pairing probe spot to the basic sequence of a specimen nucleic acid without variation, a mismatched probe spot with a mismatch of a base, and a mismatched probe spot with a mismatch of two bases or more, a second step carrying out a hybridizing reaction of the array and the specimen, and step detecting the signal from the spot of the array obtained by the step 2 by using a detecting device which detects the signal as the sum of signals from a plurality of spots in the region including a plurality of spots on the array, and setting a plurality of above regions as capable of obtaining a relation of specific signal, and determining the presence of variation by comparing result of the step 2 of the object to be measured with the result of the step 2 of the specimen without variation. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a mutation detection method and a mutation detection system useful for screening of gene mutations and the like.

  One method for determining the sequence of a substance such as a nucleic acid or checking the sequence is to use a DNA array. USP 5445934 discloses a DNA array in which 100,000 or more oligonucleotide probes are bound to 1 inch square. Such a DNA array has an advantage that multiple items can be examined at a time with a small amount of specimen. When a fluorescently labeled sample to be examined is run against such a DNA array placed on the chip, a DNA fragment having a sequence complementary to the probe on the chip binds to the probe to form a hybrid, and this hybrid By allowing only the portion where the body is formed to be identified by fluorescence, the sequence of the DNA fragment in the DNA sample can be elucidated.

  The Sequencing by Hybridization (SBH) method is a method for examining a base sequence using such a DNA array, and its details are described in USP5202021. In the SBH method, all possible sequences of oligonucleotides of a certain length are arranged on a substrate, and a completely complementary hybrid formed by a hybridization reaction with a sample DNA is detected. If a set of bodies is obtained, the set should be a set of sequences that are shifted by one base with respect to a certain sequence, and these are extracted for determination.

  When examining the complementarity of oligonucleotides and sample DNA, not limited to SBH, it is very difficult to determine the presence or absence of a hybrid with one type of probe for one test item. This is because the stability of the hybrid differs depending on the sequence, and there is no absolute signal value that can be determined to be completely complementary. Thus, a method of comparing and judging the signal intensity of a hybrid having a single base mismatch that is slightly weaker than that of a perfect match is shown in Science vol. 274 p610-614, 1996. In this method, a sequence in which a single base mismatch is arranged in the middle of the probe sequence of a 15-mer oligonucleotide is prepared, and the fluorescence intensity derived from the hybrid of the perfect match and the single base mismatch is compared, and the perfect match is stronger. If it is stronger than the ratio, it is judged as positive.

Further, USP 5733729 describes a method using a computer to know the base sequence of a specimen from a comparison of fluorescence intensity of the obtained hybrid in order to make a more accurate determination.
US Pat. No. 5,445,934 US Pat. No. 5,202,231 US Pat. No. 5,733,729 Science vol.274 p610-614, 1996

  However, the strength of binding in each hybrid actually varies depending on the GC content in each base sequence, and the strength difference between a perfect match sequence and a one-base mismatch sequence varies considerably depending on the sequence. Therefore, using the 15-mer oligonucleotide as described above and comparing with other three types of probes having a single base mismatch in the middle to determine whether or not it is a perfect match, More accuracy can be obtained by evaluating theoretically or empirically and comparing the result.

  In addition, accurate determination of signals is necessary for these accurate determinations, and usually a precise device such as a confocal laser microscope is required. Furthermore, in order to determine the intensity of the hybrid body for each probe and analyze the value to determine and determine the gene sequence, in addition to a detection device that reads the array, a larger computer device is required. It becomes an obstacle when everyone uses a DNA array easily.

  On the other hand, genetic diagnosis using such a DNA array can also be used for mass screening, individual genetic testing, or gene multisystem research. For the purpose of quickly and inexpensively processing a large amount of specimens such as searching for specimens that are being performed, the above-described precise measurement and analysis are not necessarily required. Moreover, the precise apparatus and analysis as described above are expensive. Therefore, the idea of screening for the presence or absence of mutation and conducting a detailed test only when the mutation is suspected as a result of the screening saves both cost and time.

  The present invention has been made in view of such problems, and the object of the present invention is a method suitable for mass screening that quickly determines only the presence or absence of a gene mutation without requiring an expensive apparatus and complicated analysis. Is to provide.

  The present invention relates to a probe sequence group that gives a strong signal when a hybrid is formed with a normal gene when the base sequence of a gene that has already been mutated with a normal gene is determined by information on the gene, Provided is a DNA array in which a group of probes having a sequence expected to form a hybrid is arranged in separate regions. In addition, the above object is achieved by providing a detection method using such an array.

The first aspect of the method for determining the presence or absence of a mutation in the sample nucleic acid of the present invention comprises:
A method for determining the presence or absence of mutations in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) preparing an array in which a perfectly matched probe spot, a 1-base mismatch probe spot, and a mismatch probe spot of 2 bases or more are arranged on a solid phase with no mutation on the base sequence of the sample nucleic acid; ,
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. And a process of
Having
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
The second of the region includes the mismatch probe of 2 bases or more, does not include the perfectly matched probe and the 1 base mismatch probe;
Each area contains the same number of spots,
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid without mutation under the predetermined condition is maximized in the first region and substantially zero in the second region. And
The hybridization reaction in the step (ii) is performed under the predetermined condition, and the step (iii) further includes the step of calculating the signal intensity of each region of the array obtained in the step (ii) with the DNA. Compare the signal intensity of each region obtained as a result of hybridizing the array and the sample nucleic acid without mutation under the above specified conditions, and determine the presence or absence of mutation in the sample nucleic acid based on the change in signal intensity in each region Including the step of:

The second aspect of the method for determining the presence or absence of a mutation in the sample nucleic acid of the present invention comprises:
A method for detecting the presence or absence of a mutation in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) An array in which a perfectly matched probe spot, a 1-base mismatch probe spot, a 2-base mismatch probe spot, and a mismatch probe spot of 3 bases or more are arranged on a solid phase with respect to the base sequence of the sample nucleic acid without mutation. A process of preparing
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. Having a process of
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
Including the 1 base mismatch probe spot in the second of the region, not including the perfectly matched probe,
Including the 2-base mismatch probe spot in the third of the region, not including the perfectly matched probe spot;
The fourth of the region includes the mismatch probe of 3 bases or more and does not include the perfectly matched probe spot and the 1 base mismatch probe spot,
Each area contains the same number of spots,
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid having no mutation under the predetermined condition is maximum in the first region and substantially zero in the fourth region. Is,
The hybridization reaction in the step (ii) is performed under the predetermined conditions, and the step (iii) further includes the signal intensity of each region of the array obtained by the step (ii). The array nucleic acid is compared with the signal intensity of each region of the array obtained as a result of the hybridization reaction between the array and the sample nucleic acid having no mutation under the above-mentioned predetermined conditions, and the variation in the sample nucleic acid based on the change in the signal intensity in each region Including a step of determining the presence or absence of.

The third aspect of the method for determining the presence or absence of a mutation in the sample nucleic acid of the present invention comprises:
A method for determining the presence or absence of mutations in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) preparing an array in which a perfectly matched probe spot, a 1-base mismatch probe spot, and a mismatch probe spot of 2 bases or more are arranged on a solid phase with no mutation on the base sequence of the sample nucleic acid; ,
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. Having a process of
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
The second of the region includes the mismatch probe of 2 bases or more, does not include the perfectly matched probe and the 1 base mismatch probe;
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid without mutation under the predetermined condition is maximized in the first region and substantially zero in the second region. And
The hybridization reaction in the step (ii) is carried out under the predetermined conditions, and the step (iii) further comprises a reaction with the sample nucleic acid having no mutation in the array obtained in the step (ii). Mutation due to the difference between the signal intensity relationship between the first region and the second region obtained as a result and the signal intensity relationship between the first region and the second region obtained as a result of the reaction with the sample nucleic acid Including a step of determining the presence or absence of.

  ADVANTAGE OF THE INVENTION According to this invention, an expensive apparatus and a complicated analysis are not required, but the method suitable for the mass screening which determines rapidly only the presence or absence of the variation | mutation of a gene can be provided.

Examples of techniques related to the method for determining the presence or absence of a mutation in a sample nucleic acid of the present invention include the following embodiments.
(1) A mutation determination method for monitoring a mutation in a sample nucleic acid, wherein the probe is completely complementary to a detected portion of the sample nucleic acid and unpaired with the detected portion Preparing a DNA array substrate in which a plurality of types of probes including a plurality of types of unpaired probes each having at least one combined base are distributed and fixed in a plurality of predetermined divided regions on the substrate; and A step of reacting a sample nucleic acid with a probe on a DNA array substrate; and a step of measuring a signal based on formation of a hybrid of any one of the probes and the sample nucleic acid as a total signal for each of the plurality of divided regions; And determining a mutation in the nucleic acid of the specimen from a pattern of changes in all signals for each of the plurality of divided regions.
(2) The method according to (1) above, wherein the total signal for each of the divided regions is a total light amount from one entire divided region by light emission based on the formation of the hybrid body.
(3) The method according to (2) above, wherein the light emission is fluorescence light emission.
(4) The method according to (2) above, wherein the luminescence is chemiluminescence.
(5) Using the plurality of types of probes, a segmented region containing at least one type of probe that can give a strong signal when reacted with a normal sample nucleic acid, and at least a type that can give a weak signal when reacted with a normal sample nucleic acid A group of segmented regions in which a segmented region including one type of probe and a segmented region including at least one type of probe that does not form a hybrid when reacted with a normal sample nucleic acid are independently arranged in a predetermined sequence on the substrate Prepared by reacting a normal sample nucleic acid to the segmented region group, measuring all signals for each segmented region to be measured in the segmented region group, and measuring all signals according to the sequence of the segmented region to be measured. A step of obtaining a first change pattern in advance, and reacting a sample nucleic acid with a group of divided regions of the DNA substrate to obtain a total signal for each measurement target divided region in the group of divided regions And obtaining a second change pattern of all signals according to the sequence of the measurement target segmented region, comparing the first change pattern and the second change pattern, And determining the presence or absence of a mutation. The method according to (1) above,
(6) The measurement target segmented region is a segmented region where the total signal is the strongest when reacting with a normal sample nucleic acid, and the segmented region where the total signal is the weakest (including the case where there is no signal). the method of.
(7) When each divided region constituting the divided region group reacts with the normal sample nucleic acid in the relative movement direction of the detection unit of the signal measurement device with respect to the normal sample nucleic acid, the total signal The method according to the above item (5), wherein the strengths of the components are arranged so that the strengths of the components become weaker in order.
(8) A segmented region in which the total signal is zero when reacted with normal sample nucleic acid is provided in the segmented region group, and the total signal measured by the reaction with the sample nucleic acid is measured in the segmented region, and the signal is detected. The method according to (5) above, wherein there is a mutation in the case of
(9) The above-described segmented region having the strongest total signal when reacted with the normal sample nucleic acid includes a perfectly matched probe with the normal sample nucleic acid and a probe with one base that is unpaired with the normal sample nucleic acid. The method according to item (5) or (6).
(10) The method according to (5) or (6) above, wherein the detection of all signals is performed by an area sensor.
(11) The method according to (7) above, wherein the detection of all signals is performed by a line sensor.
(12) The method according to any one of (1) to (11) above, wherein the plurality of types of probes have a length of 8 mer to 30 mer.
(13) The method according to (12) above, wherein the plurality of types of probes have a length of 12 mer to 25 mer.
(14) A DNA array substrate for monitoring a mutation in a nucleic acid of a sample, and a perfectly paired probe that is completely complementary to the sample nucleic acid on the substrate and unpaired with the sample A segmented region in which a plurality of types of probes comprising a plurality of types of unpaired probes having at least one base are arranged, and at least one type of probe that gives a strong signal when reacted with a normal base sequence And at least one of a divided region where at least one kind of probe that gives a weak signal and a divided region where at least one type of probe that doesn't give a signal are arranged are each independently arranged, DNA array substrate.
(15) The DNA array substrate according to (14), wherein the signal is fluorescence.
(16) The DNA array substrate according to (14), wherein the signal is chemiluminescence.
(17) The DNA array substrate according to (14) above, wherein the segmented region that gives a strong signal includes a perfectly paired probe and an unpaired probe having one unpaired base for the specimen.
(18) The above-described (14), wherein the plurality of divided regions are arranged such that the intensity of all signals decreases in order from the strongest region of all signals when reacted with normal sample nucleic acids in the measurement direction of the signal measuring device. The DNA array substrate according to Item.
(19) The DNA array substrate according to any one of (14) to (18), wherein the plurality of types of probes have a length of 8 mer to 30 mer.
(20) The method according to (19) above, wherein the plurality of types of probes have a length of 12 mer to 25 mer.
(21) A mutation detection system comprising: the DNA array substrate according to any one of (14) to (20) above; and a signal measuring device that measures a signal from a segmented region of the DNA array substrate. .

  And this invention made | formed based on these related techniques includes the 1st-3rd aspect mentioned above.

  The present invention will be described below by taking the case where the signal from the segmented region is fluorescence as an example. However, the signal in the present invention is not limited to fluorescence, and may be a signal due to other optical emission, and further electrically. It can be anything.

  First, the strength of the binding between nucleic acid strands constituting the hybrid is governed by various factors. When a probe having a length of about 15 mer to 20 mer is used, a hybrid having a single base mismatch is selected. It is difficult to remove completely.

  For example, in the case where a hybrid emits a signal in detection by light such as fluorescence, a sequence having a two base mismatch is more stable than a single base mismatch regardless of the position, continuous or discontinuous of the two base mismatch. The signal becomes low and almost no signal is observed in the 3 base mismatched form. However, a single-base mismatch can have a strength that is about 50% of a perfect match. In the case of normal nucleic acid, the amount of light that stands out in the region where a perfectly matched probe (perfectly matched probe) and a mismatched probe (one-base mismatched probe) with one unpaired base is placed is shown. On the other hand, in the region where the stability of the hybrid is low, the total light quantity is almost zero. On the other hand, a probe in which a nucleic acid having a mutation is completely complementary exhibits a mismatch with a probe corresponding to a normal nucleic acid, and the fluorescence intensity of a segmented region where high fluorescence is expected in the normal nucleic acid is weakened. In addition, fluorescence derived from a perfect match or a single base mismatch appears in a segmented region where the signal is expected to be low in the reaction with normal nucleic acid. Therefore, it is possible to distinguish normal nucleic acids and mutant nucleic acids by comparing the fluorescence distributions of the divided regions.

  As one means for performing such a determination with higher accuracy, the present invention performs a hybridization reaction with each probe using normal nucleic acids, and based on the fluorescence intensity of each segmented region obtained as a result, a strong intensity is obtained. Are divided into a segmented region having a probe group that gives a fluorescence, a segmented region having a probe group that does not give fluorescence, and a segmented region having a probe group having an intermediate fluorescence intensity that emits weak fluorescence, and these are arranged on a substrate in a predetermined arrangement The DNA substrate arranged in (1) is used.

  When testing multiple items at the same time, evaluate the fluorescence intensity of perfect match and mismatch for each item of probe, integrate them all, order the intensities, sort them into the above groups and arrange them. Becomes important. By arranging the segmented regions in a predetermined sequence in this way, it is possible to obtain a pattern of changes in all signals according to this sequence, specify the pattern of changes obtained with normal nucleic acids in advance, Mutation can be detected by comparing the pattern of changes obtained with a certain nucleic acid.

  The arrangement can be determined by the type of sensor. For example, if each segmented region is arranged from the left end of the substrate in the order of strong fluorescence intensity when reacted with normal nucleic acid, the fluorescence intensity is maximum at the left end and gradually when detected using a line sensor. And the zero region continues as it approaches the right edge of the substrate. In addition, when the area is evaluated by an area sensor, it is necessary to evaluate the amount of fluorescent light in at least two places: a divided area having a probe group that gives maximum fluorescence and a divided area that has a probe group that does not give fluorescence. .

  This will be described more specifically.

  In the present invention, for example, a partitioned region including a probe that forms a hybrid with a normal nucleic acid on a DNA array and a partitioned region that includes a probe that forms a hybrid with a mutant gene are set independently, and the probe corresponds to the normal nucleic acid. A method of determining whether or not it is normal by taking the ratio of the signal from the hybrid with the group and the signal from the hybrid with the mutant gene is used.

  However, in fact, if only a probe that differs from normal nucleic acid by only one base is selected as a mutant nucleic acid, the ratio is only about twice, and even if some mutant genes are included, the change in the total light amount is small and it is difficult to judge. is there.

  Therefore, in order to perform a more accurate determination, in the present invention, a probe that is a complete match sequence and a single-base mismatch sequence is specified on a DNA array substrate with respect to normal nucleic acid sequences that are shown by most specimens in mass screening. Collect and place in the area. Furthermore, it is preferable to arrange a probe exhibiting low stability such as 2-base mismatch or 3-base mismatch in a region different from the region exhibiting high stability. Then, a hybridization reaction is performed using the DNA array substrate arranged in this way, the total amount of signal is measured for each divided region, and the measurement pattern from the nucleic acid as the detection target is compared with the measurement pattern obtained with the normal nucleic acid. And the presence or absence of mutation can be tested.

  For example, in the case of 64 types of sequences (4 × 4 × 4 = 64) in which different bases are arranged at three positions in an 18-base probe (Table 1: Each sequence has a 5 ′ end on the left side and a 3 ′ end on the right side. ), There should be exactly one sequence that matches any sample. Nine types have one base mismatch, 27 types have two base mismatches, and the remaining 27 types have three base mismatches. It should be. It is relatively easy to set conditions for the hybridization reaction such that fluorescence is observed for the perfect match sequence and the one-base mismatch sequence, and no fluorescence is observed for the three-base mismatch. There are two base mismatches that may or may not form a hybrid depending on the sequence.

  If these 64 probes are grouped every 8 probes from the strongest, the fluorescence intensity of the first group is extremely strong, and the total fluorescence of the sixth, seventh and eighth groups should be zero. It is. Such classification by fluorescence intensity may be performed empirically or theoretically by calculation.

  Next, a hybridization reaction is performed on the actual system, and the total amount of fluorescence for each classified group is measured. In particular, the fluorescence amounts of the sixth, seventh and eighth groups are important. In these groups, it is expected that the amount of fluorescence is not detected. However, if fluorescence is detected contrary to the expectation, it is understood that the specimen is not normal and has a mutation in a part of the sequence. Many cases where no fluorescence is detected are normal sequences.

  However, it is necessary to compare the fluorescence amount of the first group with a normal case for more accuracy. A base mutation is suspected if it is clearly lower than expected from the normal sequence hybridization reaction. Furthermore, the region having the intermediate fluorescence intensity, that is, the fluorescence intensity distribution (fluorescence change pattern) of the second, third, and fourth groups must be compared with a normal one.

  As a more effective mutation detection method of the above method, the probes are arranged in order from the highest signal intensity to the lowest, such as perfect match from one end, one base mismatch, two base mismatch, and three base mismatch. A method can be considered in which the signal is zero in the region opposite to the region where the perfect match probe is disposed. In this case, the entire fluorescence intensity distribution pattern is a criterion for determination. For example, when the arrangement of the segment areas shown in FIG. 1A (one vertical column is one segment area) is used, a normal decrease pattern as shown in FIG. If there is a mutation, the pattern changes to have a peak in the middle.

  The above arrangement method is the same even when the multi-item inspection probes are arranged on the same substrate. All items are arranged in the order in which the fluorescence intensity is expected to be strong, such as a perfectly matched probe sequence, 1 base mismatch sequence, and 2 base mismatch sequence.

  Such a concept is not limited to the above-described method for simply evaluating mutations for three bases, and can be universally applied without limitation on the number of mutations.

  In addition, although the case where a signal was obtained when a hybrid was formed between a normal nucleic acid and a probe was explained, depending on the method for generating the signal, no signal could be obtained when the hybrid was formed, and the hybrid If no is formed, a signal may be obtained.

  As a nucleic acid as a specimen, a part necessary for detection can be taken out from a gene as a test object as necessary. Furthermore, a normal nucleic acid used as a control can be synthesized from a known sequence and used.

  The length of the probe fixed on the substrate may be a length suitable for detection, for example, preferably in the range of 8 mer to 30 mer, and more preferably in the range of 12 mer to 25 mer.

  The probe can be fixed to the substrate by various methods. However, in order to arrange the spot fixed by the probe with high density and efficiency at high speed, it is preferable to use droplet application by an inkjet method. . In addition, the amount of the probe provided in the segmented region is preferably 70 to 100 μm for each spot, and the interval is preferably set so that the spots are not connected.

  The number of spots is set considering the configuration of the sensor so that only spots where the fluorescence intensity is expected to be strong can be measured collectively, and spots where no fluorescence is expected can be measured all at once.

  The mutation detection system of the present invention includes a DNA array substrate in which a plurality of segmented regions are arranged in a predetermined sequence, and a signal measuring device in the segmented region to be measured. In the determination of the presence or absence of mutation using a DNA array substrate provided with a plurality of segmented regions, a method of detecting signals from all segmented regions and using the results for the determination of the presence or absence of mutations, A simple method for detecting signals from a part of the selected segmented regions so that the presence or absence can be detected can be used.

  In this detection system, connected to a computer, the measured values of all signals from each segmented region according to the sequence of the segmented regions are analyzed by a computer, and a pattern of changes in all signals is created and stored. By comparing the pattern of change from the normal nucleic acid and the pattern of change from the specimen, it is possible to output the determination of the possibility of mutation.

  As a sensor for signal detection, various photodiodes (for example, manufactured by Hamamatsu Photonics Co., Ltd.), for example, a split silicon photodiode array, can be used as various line sensors and area sensors. Those having a size of 1 to 2 mm × (2 to 3) mm are preferably used.

Hereinafter, the present invention will be described in more detail with reference to examples. In the following, “%” represents “% by mass”.
Example 1
Preparation of DNA array for detecting mutant genes (for line sensors)
1) Preparation of DNA array in which 64 types of probes are bound (1) Probe design The frequency of mutations in the base sequence CGGAGG corresponding to the 248th and 249th amino acid sequences of the p53 gene which is a tumor suppressor gene It is known that the highest is the case where the first C is mutated to T, the second A is mutated to G, and the third 249 amino acid G is mutated to T. Therefore, 64 types of probes were designed by paying attention to these three base sequences.

  That is, the length of the probe is 18 mer, 6 bases containing this mutation are located in the middle, and the structure before and after is sandwiched by common sequences, and is 5′-ATGAACNNNGAGNCCCAT-3 ′. Here, the portion represented by N corresponds to A, G, C, and T, which are four types of nucleobases. Since the probe DNA is a sequence complementary to the sequence to be detected (the above sequence), it is actually 5′-GATGGGNCTCNNNGTTCAT-3 ′.

FIG. 2 shows the layout of 64 types of DNA probes on the DNA array. 5′-ATGAACCGGAGGCCCCAT-3 ′, which is a sequence corresponding to a normal gene, forms a hybrid with the probe DNA 5′-GATGGGCCTCCGGTTCAT-3 ′ located in the third column from the right and the third row from the top. There is expected.
(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. Thereafter, ultrasonic cleaning was performed in a 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 1N sodium hydroxide solution that had been heated in advance for 10 minutes. Subsequently, washing with water and washing with distilled water were performed.

(surface treatment)
It was immersed in a 1% silane coupling agent aqueous solution (manufactured by Shin-Etsu Chemical Co., Ltd., trade name KBM603) at room temperature for 20 minutes, and then nitrogen gas was blown on both sides to remove moisture and dry. The silane coupling treatment was completed by baking for 1 hour in an oven heated to 120 ° C. Subsequently, 2.7 mg of EMCS (N- (6-Maleimidecaproxy) 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) Binding reaction of DNA to substrate (synthesis of 64 kinds of DNA probes)
Sixty-four probe single-stranded DNAs (described in Table 1 above) having an SH group (thiol group) at the 5 ′ end were synthesized by a conventional method.

(Discharge of DNA probe)
Dissolve each probe DNA in water and use SG Clear (aqueous solution containing glycerin 7.5%, urea 7.5%, thiodiglycol 7.5%, acetylenol EH 1%) to a final concentration of 8 μM. Diluted.

  A nozzle of a head BC62 for BJ printer (manufactured by Canon Inc.) modified so that a small amount of sample can be discharged was filled with 100 μl of the probe DNA solution. Two heads are used so that 6 types of DNA can be discharged per head, 12 types of DNA are discharged at a time, the heads are changed 6 times, and each spot of 64 types of DNA is formed independently It was made to discharge. In this way, a DNA array in which predetermined segmented regions were arranged in a predetermined sequence was obtained. The pitch of each spot including the probe array at this time was 200 μm. Further, the area where the 8 × 8 spot was formed was about 2 mm × 2 mm.

Thereafter, the substrate was left in a humidified chamber for 30 minutes, and a reaction for binding and immobilizing the probe DNA to the substrate was performed to obtain a DNA array substrate.
2) Measurement of fluorescence intensity of 64 hybrids using normal p53 sequence (1) Hybridization reaction (blocking reaction)
The DNA array substrate was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the DNA solution with no glass surface immobilized was completely washed away. Then, it was immersed in a 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours to perform a blocking reaction.
(Synthesis of model sample DNA)
A labeled DNA having the normal sequence of p53 gene (complementary to No. 42) and the same length as the probe DNA in the same length. 65 (single strand) was produced. The sequence is as shown below, and rhodamine (Rho) is bound to the 5 ′ end.
No. 65: 5′-Rho-ATGAACCGGAGGCCCCATC-3 ′
(Hybridization reaction conditions)
Place 2 ml of 50 nM model sample DNA solution containing 100 mM NaCl in a container for hybridization reaction containing a DNA array substrate, first heat at 70 ° C. for 30 minutes, then lower the temperature of the incubator to 40 ° C. and let it react for 3 hours. It was.
(2) Detection (method)
Detection was performed by connecting an image analysis processing device ARGUS (manufactured by Hamamatsu Photonics) to a fluorescence microscope (manufactured by Nikon).
(3) Results The distribution of the obtained fluorescence amount on the substrate is shown in FIG.

Example 2
Preparation of DNA array for detecting mutant genes (for line sensors)
(1) Production of mutation detection DNA array Based on the above results, Tables 2 and 3 show that these 64 types of probes are grouped every 8 probes from the strongest. The fluorescence intensity of the first group is extremely strong, and the total fluorescence amount of the sixth, seventh and eighth groups should be zero.

Next, the substrate was divided into eight lines, and the first group and the second group were arranged in descending order from the left side, and the eighth group was arranged at the right end. Next, in the same manner as in Example 1, the probes were arranged at the corresponding probe number positions using the inkjet method as shown in FIG. 4 to prepare a mutation detection DNA array. The lines formed by each group are 200 μm wide.
(2) Examination with normal gene using DNA array for mutation detection Hybridization reaction was carried out under the same conditions as in Example 1. Then, the total light quantity for each group was detected using a line sensor (S272102; manufactured by Hamamatsu Photonics).

The results are shown in FIGS. The light intensity in the first group is maximum, and in the second group, the intensity is reduced to less than half. In the sixth, seventh and eighth groups, almost no fluorescence is observed.
(Example 3)
(Detection of mutant gene using DNA array for mutant gene detection (1))
1. Synthesis of Mutation Model Specimen DNA Labeled DNA having a sequence different from the normal sequence of the p53 gene by one base in the probe region (complementary to No. 46) and having the same length as the probe 66 was produced. The sequence is as shown below, and rhodamine is bound to the 5 'end. The underlined part is the mutation.
No. 66: 5′-Rbo-ATGAACC A GAGGCCCATC-3 ′
(Hybridization reaction)
A hybridization reaction was carried out in the same manner as in Example 1. The concentration of the model sample DNA is 50 nM.
(Detection by line sensor)
As in Example 2, the array substrate after the hybridization reaction was evaluated by a line sensor. As shown in FIG. 6, fluorescence was observed in the sixth, seventh and eighth groups in which no fluorescence was observed in the case of a normal gene, and it was easily determined that this sample gene was not normal. In addition, since the fluorescence intensity in the first group and the fourth group (probe numbers 35-48) is high, it is presumed that there is a mutation in the upper right region in FIG. 2 in a sequence very close to the normal gene. .

  From this result, it was shown that the mutant gene screening method of the present invention is extremely effective.

Example 4
(Detection of mutant gene using DNA array for mutant gene detection (2))
The concentration of the model target gene used for the hybridization reaction was changed to 5 nM, and the hybridization reaction was performed under the same conditions as in Example 3. The results are shown in FIG.

  The same results as in Example 3 were obtained, and it was shown that the method of the present invention was established regardless of the hybridization reaction conditions.

Example 5
(Detection of mutant gene using DNA array for mutant gene detection (3))
Synthesis of mutation model specimen DNA Labeled DNA No. 10 having a sequence different from the normal sequence of the p53 gene by 1 base in the probe region (complementary to No. 10) and the same length as the probe 67 was produced. The sequence is as shown below, and rhodamine is bound to the 5 'end. The underlined part is the mutation.
No. 67: 5′-Rho-ATGAACCGGAG T CCCATC-3 ′
(Hybridization reaction)
A hybridization reaction was performed in the same manner as in Example 1 using the mutation model sample DNA. The analyte concentration is 50 nM. The result is shown in FIG.

  Fluorescence was observed in groups 6 and 7 where no fluorescence was observed in the normal gene, and it was found that the gene was not a normal gene. The fluorescence shows a maximum value in the second group rather than the first group showing a high value in the normal gene, and it is estimated that the probe sequence region (upper left region in FIG. 2) included in the second group has a mutation.

  This result indicates that the mutant gene screening method of the present invention is possible regardless of the type of mutation.

Example 6
(Preparation of DNA array for area sensor for mutant gene detection)
Probes divided into groups as shown in Example 2 were arranged in a divided area on the substrate instead of in a line form, and a probe solution was discharged in the arrangement shown in FIG. 9 to prepare a DNA array.

  Next, hybridization reaction was performed using the mutation model sample used in Example 4. As a result, fluorescence was observed in groups 6, 7, and 8 where no fluorescence was observed in the normal gene, and it was easily determined that this gene was a mutant gene.

(A) is a figure which shows an example of arrangement | positioning of the division area on a DNA array board | substrate, (b) is the fluorescence corresponding to arrangement | positioning of the division area obtained when a line sensor is used in the measurement using this board | substrate. It is a figure which shows intensity distribution (pattern). It is an arrangement plan of 64 kinds of DNA probes. It is a figure which shows the result of fluorescence intensity. It is a figure which shows the result of fluorescence intensity, Gr. No. Represents a group number. It is a figure which shows the fluorescence intensity distribution (pattern) obtained corresponding to arrangement | positioning of the division area measured when a line sensor is used. It is a figure which shows the fluorescence intensity distribution (pattern) obtained corresponding to arrangement | positioning of the division area measured when a line sensor is used. It is a figure which shows the fluorescence intensity distribution (pattern) obtained corresponding to arrangement | positioning of the division area measured when a line sensor is used. It is a figure which shows the fluorescence intensity distribution (pattern) obtained corresponding to arrangement | positioning of the division area measured when a line sensor is used. It is a figure which shows the result of fluorescence intensity, Gr. No. Represents a group number.

Claims (9)

A method for determining the presence or absence of mutations in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) preparing an array in which a perfectly matched probe spot, a 1-base mismatch probe spot, and a mismatch probe spot of 2 bases or more are arranged on a solid phase with no mutation on the base sequence of the sample nucleic acid; ,
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. And a process of
Having
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
The second of the region includes the mismatch probe of 2 bases or more, does not include the perfectly matched probe and the 1 base mismatch probe;
Each area contains the same number of spots,
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid without mutation under the predetermined condition is maximized in the first region and substantially zero in the second region. And
The hybridization reaction in the step (ii) is performed under the predetermined condition, and the step (iii) further includes the step of calculating the signal intensity of each region of the array obtained in the step (ii) with the DNA. Compare the signal intensity of each region obtained as a result of hybridizing the array and the sample nucleic acid without mutation under the above specified conditions, and determine the presence or absence of mutation in the sample nucleic acid based on the change in signal intensity in each region A method for determining the presence or absence of a mutation in a sample nucleic acid, comprising the step of:   The method of claim 1, wherein the signal from the spot is light.   The method according to claim 2, wherein the detection device is an area sensor or a line sensor. A method for detecting the presence or absence of a mutation in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) An array in which a perfectly matched probe spot, a 1-base mismatch probe spot, a 2-base mismatch probe spot, and a mismatch probe spot of 3 bases or more are arranged on a solid phase with respect to the base sequence of the sample nucleic acid without mutation. A process of preparing
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. Having a process of
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
Including the 1 base mismatch probe spot in the second of the region, not including the perfectly matched probe,
Including the 2-base mismatch probe spot in the third of the region, not including the perfectly matched probe spot;
The fourth of the region includes the mismatch probe of 3 bases or more and does not include the perfectly matched probe spot and the 1 base mismatch probe spot,
Each area contains the same number of spots,
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid having no mutation under the predetermined condition is maximum in the first region and substantially zero in the fourth region. Is,
The hybridization reaction in the step (ii) is performed under the predetermined conditions, and the step (iii) further includes the signal intensity of each region of the array obtained by the step (ii). The array nucleic acid is compared with the signal intensity of each region of the array obtained as a result of the hybridization reaction between the array and the sample nucleic acid having no mutation under the above-mentioned predetermined conditions, and the variation in the sample nucleic acid based on the change in the signal intensity in each region A method for determining the presence or absence of a mutation in a sample nucleic acid, comprising the step of determining the presence or absence of a nucleic acid.   The method of claim 4, wherein the signal from the spot is light.   The method according to claim 5, wherein the detection device is an area sensor or a line sensor. A method for determining the presence or absence of mutations in a sample nucleic acid using an array in which a plurality of probe spots are arranged on a solid phase,
(I) preparing an array in which a perfectly matched probe spot, a 1-base mismatch probe spot, and a mismatch probe spot of 2 bases or more are arranged on a solid phase with no mutation on the base sequence of the sample nucleic acid; ,
(Ii) a step of causing a hybridization reaction between the array and a specimen;
(Iii) Detection using a detection device that detects signals from the spots of the array obtained in the step (ii) as a sum of signals from the plurality of spots in an area including the plurality of spots on the array. Having a process of
The array comprises a plurality of the regions,
Including the perfectly matched probe spot in the first of the region;
The second of the region includes the mismatch probe of 2 bases or more, does not include the perfectly matched probe and the 1 base mismatch probe;
Each of the spots in each region and the labeled nucleic acid sample without mutation when the signal intensity of each region is defined as the sum of the signals indicated by each of the spots in each region The signal intensity obtained from each region as a result of hybridizing the spot and the sample nucleic acid without mutation under the predetermined condition is maximized in the first region and substantially zero in the second region. And
The hybridization reaction in the step (ii) is carried out under the predetermined conditions, and the step (iii) further comprises a reaction with the sample nucleic acid having no mutation in the array obtained in the step (ii). Mutation due to the difference between the signal intensity relationship between the first region and the second region obtained as a result and the signal intensity relationship between the first region and the second region obtained as a result of the reaction with the sample nucleic acid A method for determining the presence or absence of a mutation in a sample nucleic acid, comprising the step of determining the presence or absence of a nucleic acid.   The method of claim 7, wherein the signal from the spot is light.   The method according to claim 8, wherein the detection device is an area sensor or a line sensor.

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