JP2007006723A - Nucleic acid microarray and method for detecting nucleic acid therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid microarray which can be used to detect the expression of a gene or the like without not depending to the manual skill of an experimenter, and to provide a method for detecting a nucleic acid therewith. <P>SOLUTION: This nucleic acid microarray containing washing level meter probes, and the method for detecting the nucleic acid is characterized by comprising the following processes (1) to (4): (1) a process for preparing a sample nucleic acid, (2) a process for bringing the sample into contact with the microarray to perform a hybridization reaction, (3) a process for stepwisely washing the microarray after the hybridization reaction from the weak side of washing degree to the strong side, and (4) a process for detecting the hybrid formed in (4). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid microarray and a nucleic acid detection method using the same.

  Nucleic acid microarrays are obtained by independently immobilizing a large number of nucleic acid probes on a carrier. As the nucleic acid probe, cDNA or synthetic oligonucleic acid is often used. Conventionally, a nucleic acid probe immobilized on a surface-treated glass or silicon carrier has been used. In recent years, the carrier has been modified by other methods such as a gel carrier.

  A nucleic acid detection method using a hybridization technique is a method in which a nucleic acid sample to be tested is hybridized in a sequence-specific manner to a nucleic acid probe immobilized on a nucleic acid microarray, and the hybrid formed in a sequence-specific manner is a fluorescent substance. This is a method for quantitatively or qualitatively examining molecules containing a nucleobase sequence in a sample corresponding to a plurality of nucleic acid probes. This method is used for analyzing the expression level of a plurality of nucleobase sequences or the sequence itself of a specific nucleobase sequence.

  When detecting a nucleic acid, conventionally, a hybridization reaction is carried out under appropriate conditions set in advance, and then a nucleic acid sample remaining on the surface of the array or other unnecessary substances is removed by a washing operation, which is specific to the nucleic acid probe. A nucleic acid sample forming a hybrid is detected. Nucleic acid probes are often designed to be complementary or identical to a desired nucleobase sequence to be detected, such as sequence analysis and functional analysis. As the nucleic acid probe, a relatively long chain nucleic acid such as cDNA, a short synthetic oligonucleic acid or the like is used. When using synthetic oligonucleic acid as a nucleic acid probe, for organisms such as humans and mice that have accumulated knowledge of genetic information, use their base sequence information and pay attention to the homology and function of each sequence. It is possible to design a synthetic oligonucleic acid sequence and prepare a nucleic acid probe.

  Nucleic acid detection using nucleic acid microarrays is currently being automated and accelerated. On the other hand, the detection result is still often influenced by the experimenter's technique. In order to correct such experimenter's procedures, the nucleic acid microarray incorporates criteria for determining the success or failure of the experiment. This criterion is called a control probe. The control probe includes a negative control probe that corresponds to a nucleic acid sequence that should not be included in the nucleic acid sample, a positive control probe that corresponds to a nucleic acid sequence that is reliably included in the nucleic acid sample, and detection between samples. There are control probes for correcting the results, control probes for confirming the quality of the nucleic acid sample, and the like. Of these, negative control and positive control are used to confirm the success of the experimental technique itself. For example, if a signal is obtained from the negative control probe or if no signal is obtained from the positive control, the data reliability Will be doubted.

  However, it is not clear to what extent the signal should be reduced or maintained during the washing operation for the negative control and positive control signals, and furthermore, it is difficult to express the criteria as numerical values.

  Also, when specific hybrid and non-specific hybrid dissociation conditions (Tm value, salt concentration) are close, and specific hybrids are similar in specificity, they are completely divided. It was difficult to uniquely determine the cleaning conditions that can be performed.

  The present invention aims to solve the above problems.

  As a result of intensive studies to solve the above problems, the present inventors designed a probe sequence including a known specific sequence that can be correlated with the cleaning conditions, and used the probe to increase the degree of cleaning progress. The inventors have found that it can be quantified and completed the present invention.

  That is, the present invention is a nucleic acid microarray including a washing level meter probe. Moreover, it is the detection method of the nucleic acid using the array.

  According to the present invention, the washing step is quantitatively clarified when detecting the nucleic acid microarray. In addition, the possibility of including a signal derived from cross-hybridization can be estimated, and the data reliability of detection using a nucleic acid microarray is improved. Furthermore, even when the Tm values of the target probes mounted on the nucleic acid microarray vary, the degree of cleaning of each probe can be clarified.

  The first of the present invention is a nucleic acid microarray including a washing level meter probe.

The “wash level meter probe (hereinafter sometimes simply referred to as a wash probe)” is a probe used for quantifying the wash step when detecting nucleic acid using hybridization using a nucleic acid microarray or the like. Each of the washing probes fixed to the nucleic acid microarray constitutes an assembly of probes (probe group) having different temperatures required for dissociating complementary strands or partially complementary strands. Each of the probes constituting the washing probe group shows a different dissociation temperature of the double-stranded nucleic acid calculated by a Tm value calculation formula or the like. In addition, they are designed stepwise or in a differential manner near the Tm value at the same salt concentration of a probe containing a target sequence to be detected (hereinafter referred to as a target probe).

  The base composition of such a cleaning probe may be any combination of bases. Also, the similarity between the sequences of each washing probe is not a problem. However, wash probes that hybridize to sequences that are likely to be included in nucleic acid samples should not be designed. Specifically, when designing a cleaning probe, it is desirable to search each designed cleaning probe from a public database such as GenBank and design so as not to include a continuous complementary sequence of at least 16 bases or more. Moreover, those designed so that the Tm values of the respective washing probes are different depending on the chain length or the base sequence are preferable. Furthermore, it is more preferable that the cleaning probe is composed of a certain sequence as one unit and a repeating sequence of the unit. With such a probe, a group of probes having different Tm values can be easily designed by changing the number of units.

  The designed cleaning probe is prepared by a known method such as a chemical synthesis method or a method of preparing from cDNA.

  The prepared washing probe is immobilized on or in the array as a probe constituting a nucleic acid microarray (hereinafter referred to as a nucleic acid array). The nucleic acid array is an array in which a plurality of probes are independently immobilized on a carrier such as a flat substrate. A nucleic acid sample molecule containing a base sequence corresponding to the target probe immobilized on the nucleic acid array is obtained by specifically hybridizing the nucleic acid sample to be tested to the base sequence of the target probe. It can be examined quantitatively or qualitatively.

Various forms of nucleic acid arrays are known in addition to those using the planar substrate. Some of the present inventors have also developed a fiber type microarray (Genopal ^™ , http://www.mrc.co.jp/genome/top.html) that uses fibers as a carrier for immobilizing a probe. .

  The method of immobilizing the probe when producing the nucleic acid array is selected in view of the nature of the carrier.

  By mounting the above-described washing probe or washing probe group on the nucleic acid array prepared as described above, highly reproducible data that is not affected by the experimenter's procedure can be acquired.

  Next, a “nucleic acid detection method using a nucleic acid array” as the second invention will be described.

  As the nucleic acid sample, a nucleic acid having a complementary sequence to the target probe immobilized on the nucleic acid array, a nucleic acid having no complementary sequence to the target probe, or a mixture thereof can be considered. The nucleic acid sample may be of any type or origin, such as DNA or RNA. For example, it can also be prepared using PCR, reverse transcription reaction, transcription reaction and the like (see “Molecular Cloning (CSHL PRESS)”).

  Furthermore, in the present invention, the nucleic acid sample includes a nucleic acid complementary (in some cases, partially complementary) to the washing probe mounted on the nucleic acid array. These complementary nucleic acids are used as an index for determining the washing level in the washing operation described later. Further, when the original nucleic acid sample contains a nucleic acid having a sequence complementary to the washing probe, it is not necessary to prepare it separately.

  Next, the nucleic acid sample is labeled as necessary. Examples of the labeling include labeling with a fluorescent substance, a radioisotope, an enzyme, etc. (see “DNA array and latest PCR method (Shujunsha)”). In addition, a method of introducing an intercalator into a double strand in which a hybrid is formed without labeling the specimen is also used.

  Next, the above-described nucleic acid sample is brought into contact with the nucleic acid array to perform a hybridization reaction. The hybridization reaction is carried out by preparing a hybridization solution from a nucleic acid sample and a predetermined reagent and bringing it into contact with a nucleic acid array (see “DNA Array and Newest PCR Method (Shujun)”).

  Here, “contact” means a state in which the probes fixed to the nucleic acid array are in contact with the nucleic acid sample. The method includes immersing the nucleic acid array in a nucleic acid solution, or a probe of the nucleic acid array. Examples thereof include a method of allowing a nucleic acid sample to pass through or stay in a fixed site.

  In the hybridization reaction, it is important to set conditions for the hybridization temperature, the salt concentration of the hybridization solution, and the pH of the solution. These conditions are sufficient for a specific nucleic acid complementary to the target probe contained in the nucleic acid sample to form a hybrid with the probe with respect to the base sequence of the probe fixed to the nucleic acid array. It is preferable that More preferably, the conditions are such that the specific nucleic acid sample can form a hybrid with respect to the target probe in preference to the non-specific nucleic acid sample.

  The above conditions can be determined by calculating the Tm value of each probe in consideration of probe sequence information, hybridization reaction conditions, and the like, and using the Tm value as an index. The Tm value can be calculated by, for example, a calculation formula proposed by Baldino F.J et al.

  Next, a cleaning operation is performed. Usually, after hybridization, the nucleic acid array contains not only hybrids of specific nucleic acids and probes, but also non-specific hybrids that are non-specifically bound to the probes and enzymes that are mixed in the sample. Things exist. In order to detect only specific nucleic acid hybrids, a washing operation for removing these contaminants is required.

  The cleaning solution preferably contains a salt and a surfactant, but mainly uses SSC solution, Tris-HCl solution, Tween20 solution, SDS solution or a mixture thereof prepared from sodium chloride, sodium citrate, etc. Often to do. In addition, the cleaning liquid needs to have an appropriate salt concentration, temperature, and pH. This condition is determined using the above-described Tm value as an index.

  As the washing operation, a solution that does not contain a nucleic acid sample and is optimized at a certain salt concentration, temperature, and pH is used as a washing solution, and the nucleic acid array is immersed in the solution, the surface of the nucleic acid array or the probe binding site. A method of passing or retaining the cleaning liquid is used. For example, the cleaning liquid is injected into a temperature-controllable container, the temperature of the cleaning liquid is kept constant, and the nucleic acid array is immersed in the container.

  By the above washing operation, ideally, only the hybrid with the target probe (specific hybrid) remains on the nucleic acid array, and all non-specific hybrids, contaminants, etc. are washed away. However, if the conditions for dissociation between specific hybrid and non-specific hybrid (Tm value, salt concentration) are close, and if the specificity of specific hybrid is similar, they are completely divided. It is difficult to uniquely determine the cleaning conditions that can be performed.

  The above problem can be solved by using the “nucleic acid microarray equipped with a washing level meter probe” of the present invention.

  That is, for the washing probes mounted on the nucleic acid array, the degree of washing is gradually changed from a weaker side (washing conditions in which hybrids are less likely to dissociate) to a stronger side (conditions in which hybrids are more likely to dissociate). The washing conditions can be determined while observing the dissociation state of the hybrid with the washing probe. As described above, the washing probe is designed in stages near the Tm value of the target probe, and the nucleic acid sample contains complementary (partially complementary) nucleic acids corresponding to the washing probe. Optimal washing conditions corresponding to the probe and nucleic acid sample can be determined.

  Specifically, after washing using a washing solution having a high salt concentration and a low solution temperature, detection is performed, and the nucleic acid array after the detection has a salt concentration lower than that of the previous washing solution and / or a solution. Washing is performed using a washing solution having a high temperature, and the process of detecting again is performed stepwise and multiple times to determine the washing conditions.

  As the detection method, various detection methods can be used depending on the labeling method of the nucleic acid sample. For example, a fluorescence labeling detection method, a chemiluminescence method, a radioisotope, or the like can be used.

  In the fluorescence labeling detection method, a fluorescent signal of a nucleic acid sample labeled with a pre-labeled nucleic acid or a nucleic acid labeled with an intercalator is detected.

  As a detection apparatus, it can detect with the fluorescence microscope provided with the member suitable for detecting the fluorescence of a nucleic acid array, such as a cooling CCD camera, for example. Other detection devices such as laser scanners and X-ray films can be used.

  In the detection, in order to prevent the nucleic acid array surface or the entire surface from being dried, it is desirable to detect at least in an environment without moisture evaporation. As a method for preventing drying, for example, the nucleic acid array is immersed in a case containing a solution and detected in a liquid phase, or the nucleic acid array is enclosed in a sealed container in a saturated water vapor atmosphere without moisture evaporation. And the like. However, even if the array surface is dried, it is not necessary to prevent the array surface from drying as long as re-washing and re-detection are possible.

  Hereinafter, the present invention will be specifically described with reference to examples.

<Example 1>
In order to confirm whether the washing probe functions as expected, a nucleic acid array containing an oligonucleic acid functioning as a washing probe was prepared and a washing experiment was performed.

1. Preparation of Nucleic Acid Samples As external control nucleic acid samples to be hybridized to washing probes, Cy5 end-labeled oligonucleotides having the sequences shown in SEQ ID NOs: 1 and 2 are used as model nucleic acid samples to be hybridized to other than washing level meter probes. Cy5 end-labeled oligonucleotides having the sequences shown in Table 1 were synthesized (see Table 1). A model nucleic acid sample solution having the composition shown in Table 2 containing these Cy5 end-labeled oligonucleotides was prepared.

2. Probe synthesis The probes shown in Table 3 were synthesized. SEQ ID NOs: 5 to 10 function as washing probes.

The probe of SEQ ID NO: 11 was used as a model for cross-hybridization to contrast with SEQ ID NO: 12. The nucleic acid probe of SEQ ID NO: 12 was used as an indicator of accurate data because the sample nucleic acid of SEQ ID NO: 4 could be captured with perfect complementation.

3. Production of Nucleic Acid Array A hollow fiber bundle was produced using the arrangement fixing device shown in FIG. In the figure, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, two 0.1mm thick perforated plates (21) each having a hole of 0.32 mm in diameter and a total distance of 228 in 12 rows and 19 rows each with a distance between the centers of the holes of 0.42 mm. Got ready. These perforated plates were overlapped, and polycarbonate hollow fibers (31) (added by 1% by mass of carbon black manufactured by Mitsubishi Engineering Plastics) were passed through each of the holes one by one.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 20 mm and 100 mm from one end of the hollow fiber. That is, the interval between the two perforated plates was 80 mm.

  Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object (41). In this way, a container having an open top only was obtained. The resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the total weight of polyurethane resin adhesives (Nippon Polyurethane Industry Co., Ltd., Nippon-Poran 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle.

Next, a gel precursor polymerizable solution containing a monomer and an initiator mixed at a mass ratio shown in Table 4 was prepared for each probe mounted on the array.

  Next, in order to fill the hollow portion of the corresponding hollow fiber of the hollow fiber bundle with the gel precursor polymerizable solution containing each nucleic acid probe prepared above so as to have the probe arrangement shown in FIG. The container containing the functional solution and the hollow fiber bundle prepared above were placed in a desiccator. After the inside of the desiccator was decompressed, the unsealed end of each yarn of the hollow fiber bundle was immersed in a container containing the predetermined polymerizable solution. Nitrogen gas was sealed in a desiccator, and a gel precursor polymerizable solution containing a capture probe was introduced into the hollow portion of the hollow fiber. Subsequently, the inside of a container was 70 degreeC and the polymerization reaction was performed over 3 hours.

  In this way, a hollow fiber bundle was obtained in which the probe was held in the hollow part of the hollow fiber via the gel-like material.

  Next, the obtained hollow fiber bundle was sliced in a direction orthogonal to the longitudinal direction of the fiber using a microtome to obtain 50 thin sheet sheets (nucleic acid arrays) having a thickness of 0.25 mm.

4). 2. Hybridization reaction, washing and detection operation To the nucleic acid array prepared in 1. above. The nucleic acid sample prepared in step 1 was contacted, and hybridization was performed at 65 ° C. for 17 hours in a thermostatic bath.

After hybridization, the array was washed under the washing conditions shown in Table 5 and detected sequentially. The detection operation was carried out by immersing the array in 2 × SSC using a cooled CCD camera type nucleic acid array automatic detection apparatus, and covering the cover glass, and then measuring the fluorescence signal intensity of the labeled nucleic acid sample molecule.

5. The fluorescence signal intensity obtained as a result of signal intensity measurement is summarized in FIG. In this example, SEQ ID NOs: 5 to 10 are probe sequences used as washing level meters, and based on the calculated Tm value so that the double strands are easily dissociated sequentially from SEQ ID NO: 5. Designed. By using these signal intensity patterns as an index, it is possible to perform highly reproducible washing and obtain highly reliable data.

  In this example, the difference in signal intensity between SEQ ID NO: 6 and SEQ ID NO: 7 is 100 times or more after the sixth wash. At that time, the signal intensity of SEQ ID NO: 11, which is a pseudo-cross hybrid, is greatly reduced. However, the signal intensity of SEQ ID NO: 12, which is completely complementary, is not significantly reduced.

  That is, the measurement result of the sixth time is a result of capturing the hybridization of the completely complementary sequence and eliminating the hybridization of the non-completely complementary sequence, and indicates that the washing condition is highly reliable.

  Therefore, in order to perform this experiment with good reproducibility and accuracy, start with washing with a little weak stringency, for example, 65 ° C, about 0.5xSSC, repeat detection and rewashing, and repeat the signals of SEQ ID NOS: 6 and 7. If the difference in intensity is used as a guide and the data when the difference is 100 times or more is used, highly accurate data can be obtained with good reproducibility.

  In general, data validation of nucleic acid arrays may be equipped with a positive control as a probe that gives a clear signal intensity or a negative control that should not give a signal. It was very difficult to quantitatively capture the progress of the washing operation. As in this example, it is possible to quantitatively grasp the progress of washing by mounting a plurality of washing probes having different Tm values and adding a corresponding sample from the outside.

It is a figure of the arrangement | sequence fixing jig which manufactures a fiber array. It is a figure which shows the design of a nucleic acid array. It is the figure which showed the fluorescence signal intensity | strength of the nucleic acid array in each washing conditions.

Explanation of symbols

11... Hole 21... Perforated plate 31... Hollow fiber 41.

SEQ ID NO: 1 Synthetic DNA
SEQ ID NO: 2 synthetic DNA
SEQ ID NO: 3 synthetic DNA
SEQ ID NO: 4 synthetic DNA
SEQ ID NO: 5 synthetic DNA
SEQ ID NO: 6 synthetic DNA
SEQ ID NO: 7 synthetic DNA
SEQ ID NO: 8 synthetic DNA
SEQ ID NO: 9 synthetic DNA
SEQ ID NO: 10 synthetic DNA
SEQ ID NO: 11 synthetic DNA
SEQ ID NO: 12 synthetic DNA

Claims (5)

  Nucleic acid microarray containing wash level meter probes.   The array according to claim 1, wherein the washing level meter probe is designed stepwise near the Tm value of the target probe.   The array according to claim 1 or 2, wherein the washing level meter probe is designed so that the Tm value of each probe differs depending on the difference in chain length or base sequence.   The array according to any one of claims 1 to 3, wherein the washing level meter probe has a certain sequence as one unit and is a repeating sequence of the unit. A method for detecting a nucleic acid comprising the following steps (1) to (4):
(1) A step of preparing a sample nucleic acid.
(2) A step of bringing the sample nucleic acid into contact with the array according to any one of claims 1 to 4 and performing a hybridization reaction.
(3) A step of washing the array after the hybridization reaction step by step from the weak side to the strong side.
(4) A step of detecting the formed hybrid.

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