JP2008151647A - Confirmation method of probe position on microarray - Google Patents

Abstract

A method for obtaining positional information of probes fixed on a microarray is provided.
A probe from a signal from a target nucleic acid label detected by observing a surface on which a probe fixing region exists with a confocal microscope by a hybridization reaction step between the probe on the microarray and the labeled target nucleic acid. And the step of removing the target nucleic acid with a washing buffer, and these steps are repeated for the same microarray.
[Selection] Figure 1

Description

  The present invention relates to a method for confirming the sequence and position of a plurality of probes fixed on a nucleic acid microarray substrate.

  A nucleic acid microarray is a high-throughput method that obtains sequence information of specific DNA or RNA contained in a sample by immobilizing a plurality of nucleic acid sequences (probes) on a substrate and performing a hybridization reaction with a fluorescently labeled sample solution. It is an analysis method. The hybridization reaction has been known as a technique for reading nucleic acid sequence information because it is based on the characteristic of a base that strongly binds between adenine and thymine and between guanine and cytosine. However, since a cDNA spotted on a membrane was used in the past, the amount of nucleic acid immobilized on the membrane varied, and the hybridization reaction efficiency was low, making it difficult to conduct an accurate examination. However, in recent years, a microarray production technique using a nucleic acid extension reaction on a substrate by photodeprotection has been born by applying a photolithographic technique (SPA Fodor et al, Science 251, 767 (1991)). , S. P. A. Fodor et al, Nature 364 (6437): 555-6 (1993)). As a result, the number of probes immobilized on the substrate has increased explosively, and has attracted attention as a major device in genome science that handles vast amounts of base sequence information. In recent years, the number of probes immobilized on a microarray has increased, and techniques for mass-producing arrays having similar probes are also progressing. Therefore, it is expected to be used in a wide range of applications, such as high-speed sequencing of multiple species, application to tailor-made medicine by individual polymorphism analysis, and pathological diagnosis such as cancer and diabetes by expression level analysis. In addition to photolithographic production methods, various production methods have been developed, such as a method of applying and synthesizing on a substrate by an ink jet method, and a method of applying and immobilizing oligo DNA by ink jet. Various types of microarrays have been developed.

  There are two types of DNA immobilized on the microarray: a type in which cDNA is spotted and a type in which an oligonucleic acid of several tens of bases is synthesized or spotted on a substrate. These methods are properly used according to the purpose of use of the microarray, but in recent years, the use of oligotypes has increased. In recent years, more than tens of thousands of probes can be fixed on a single array, and microarrays with probes for all human and mouse genes can be produced. Has been.

  It is a general method of using microarrays, but the region on which the probes on the array are fixed is brought into contact with DNA (or mRNA) extracted from the specimen (fluorescently labeled) (target nucleic acid) and kept at an appropriate temperature. Incubate for several hours to perform the hybridization reaction. As a result, if there is a target nucleic acid containing a sequence complementary to the probe in the sample, a reaction with the corresponding probe occurs and the sample is fixed at that location. Only the corresponding position is observed with fluorescence. Thus, by knowing which position emits fluorescence as a result of fluorescence observation, it is possible to know what sequence DNA or mRNA was present in the specimen.

  In a microarray, multiple probes can be immobilized on a single substrate, and the corresponding sequences are identified from the positional information of the probes that emitted fluorescence during fluorescence observation. There is an advantage that can be done in parallel. Compared with conventional methods for tracking individual probe reactions, there has never been a high-throughput method for verifying a large number of reactions with a small amount of sample like a microarray.

However, in order to be able to use the microarray as a device for extracting genome information in this way, it is necessary that the fixed position of the probe and the base sequence correspond exactly.
JP-A-7-27768 JP 11-187900 A SPAFodor et al, Science251,767 (1991), SPAFodor et al, Nature364 (6437): 555-6 (1993)

  When a large number of probes are fixed, the sequence is known only from the position information of each probe. Therefore, if there is some mistake in the board creation stage, or if the reaction does not proceed as expected and the probe with the planned arrangement is not synthesized on the board, the predetermined arrangement is placed at the predetermined position. Even if it could not be fixed, it was difficult to verify it later.

  One reason is that the amount of DNA immobilized on the substrate is extremely small, so that detection itself may be difficult. Even if detection is possible, it is very difficult to specify the base sequence. Is difficult. In the case of oligo DNA or the like, since the sequence is short, it cannot be amplified by a method such as ordinary PCR.

  Another reason is that the number of probes becomes enormous. In principle, a hybridization reaction with a fluorescently labeled target nucleic acid complementary to a predetermined probe can be carried out as usual, and it can be confirmed whether the planned position is illuminated. However, when the number of probes to be confirmed increases, it is necessary to carry out a large number of hybridization reactions, and that many microarrays are used.

  Therefore, if the result of the hybridization reaction is completely contrary to expectations, does it mean a novel phenomenon that is completely different from what was expected, or simply a mistake in the probe fixing stage on the microarray side? I couldn't isolate it.

  Therefore, an object of the present invention is to provide a method for confirming that a plurality of probes each having a predetermined arrangement fixed on a microarray are fixed at a predetermined position.

  In order to solve the above-described problem, the method for obtaining the positional information of the probes fixed on the microarray according to the present invention includes a complementary sequence to the probe in a probe fixing region where a plurality of types of probes on the microarray are fixed. And a step of introducing a solution containing at least one target nucleic acid labeled and carrying out a hybridization reaction between the target nucleic acid and the probe, and after the reaction, confocal in the presence of the solution A step of observing the surface on which the probe fixing region is present with a mold microscope and confirming the position of the probe from a signal from the detected target nucleic acid label; and a step of removing the introduced target nucleic acid with a washing buffer; And the above process is repeated for the same microarray.

  As described above, according to the present invention, it is possible to confirm whether a plurality of probes fixed to a microarray on a single microarray have a predetermined arrangement is fixed at a predetermined position. In addition, this method can provide a reliable microarray.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  The microarray targeted by the present invention includes any microarray as long as the probe arrangement is specified in advance and the probes are spotted at predetermined positions in an array for each type.

  As the target nucleic acid labeling substance of the present invention, any labeling substance can be used without particular limitation as long as it can be detected by a confocal microscope system. In general, a fluorescent substance capable of highly sensitive detection is used. It can be used suitably. As fluorescent substances, for example, Cy3 and Cy5 are often used as fluorescent substances for nucleic acid labeling, and these fluorescent substances can be cited as effective fluorescent substances in the present invention. If the target nucleic acid is distinguished and labeled with two or more types of fluorescent substances having different fluorescence wavelengths, a plurality of types of probes can be detected simultaneously by a single hybridization reaction.

The method for obtaining positional information of probes on a nucleic acid microarray of the present invention is characterized in that the following steps (i) to (iii) are repeatedly performed on the same microarray.
(I) introducing a solution containing at least one type of target nucleic acid having a sequence complementary to the probe into a probe fixing region to which a plurality of types of probes are fixed on the microarray; A step of performing a hybridization reaction with the probe.
(Ii) After the hybridization reaction, in the presence of a solution containing one type of target nucleic acid, the surface on which the probe fixing region exists is observed with a confocal microscope, and the signal from the detected target nucleic acid label is detected. Confirming the position of the probe;
(Iii) A step of removing the target nucleic acid introduced in step (i) with a washing buffer.

  The hybridization reaction used in the present invention is performed in a chamber. Preferably, it has an opening surface having a width that covers the probe fixing region on the nucleic acid microarray and a cross section that can be in close contact with the microarray substrate to be used, and at least two communication ports. The opening surface of the hybridization chamber is fixed to the nucleic acid microarray so as to be in close contact with the nucleic acid microarray substrate including the probe fixing region, thereby preparing a closed space for performing the hybridization reaction of the nucleic acid microarray. Furthermore, the hybridization chamber needs to use at least an observation region of a material that can transmit light used for observation with a confocal microscope. In consideration of this point, as a material for the hybridization chamber, polycarbonate having high translucency can be preferably used.

  In the present invention, the step of performing hybridization for each probe, the step of confirming the probe position by the hybrid of the target nucleic acid and the probe with a confocal microscope, and the target nucleic acid and the probe and the hybrid remaining in the solution subjected to the hybridization reaction The probe position is confirmed by a detection cycle having a step of removing the target nucleic acid to be formed with a washing buffer. Therefore, in order to acquire position information of a plurality of types of probes fixed on the microarray, it is necessary to continuously perform a plurality of detection cycles for one microarray. In addition, the number of types of target substances that can be distinguished and detected at the same time is limited to the number of probe types that can detect probe positions in a single detection cycle. Detectable.

As one aspect of the present invention when detecting a plurality of probes, for example, when using four types of target nucleic acids using a microscope system capable of detecting three types of fluorescent dyes, the detection cycle of the present invention is changed to the following. Can be set as follows.
1) Using one kind of fluorescent dye, one detection cycle is performed for each target nucleic acid, and a total of four detection cycles are performed.
2) Using two types of fluorescent dyes, one detection cycle is performed for each of the two types of target nucleic acids, and a total of two detection cycles are performed.
3) Using three types of fluorescent dyes, one detection cycle is performed for three types of target nucleic acids, and for one remaining target nucleic acid, one detection cycle is performed using one type of fluorescent dye, for a total of 2 Perform one detection cycle.

  Within the range of types of fluorescent dyes that can be detected as described above, one probe detection cycle may be performed, one detection cycle may be performed for each target nucleic acid, or these detection cycles may be performed. You may carry out in combination.

  In addition, when multiple detection cycles are performed, the fluorescence from the target nucleic acid fluorescent label is detected by focusing on the surface to which the probe is fixed, so that the fluorescence from the hybrid target nucleic acid formed in the previous detection cycle is also detected. It will be detected at the same time. Therefore, it is necessary to dissociate and remove the target nucleic acid that forms a hybrid after each detection cycle. Therefore, a step of washing the microarray with a washing buffer at a temperature at which the hybrid of the probe and the target nucleic acid is dissociated between detection cycles is performed. In the washing step, a washing buffer is introduced into the hybridization chamber, the temperature is raised to a temperature sufficient for dissociation of the hybrid, and it is allowed to incubate for a certain period of time before being discharged, or can be maintained at this temperature. You may distribute | circulate the washing | cleaning buffer in the chamber in the range.

  In the present invention, using the characteristics of the confocal microscope, it is possible to focus and observe only the surface where the probe fixing region exists. Therefore, it is possible to detect only the signal from the label of the target nucleic acid that forms a hybrid with the probe, as distinguished from the fluorescence from the target nucleic acid that does not form a hybrid with the probe that exists in the solution in the hybridization chamber.

  FIG. 1 shows an overview of one preferred embodiment. A confocal microscope (a) (LSM510, manufactured by Carl Zeiss) is installed, and the microarray (c) is fixed to the focal portion. The hybridization chamber (b) is fixed on the array so as to cover the probe fixing region of the microarray, and sealed so that the solution does not leak. At this time, the height of the chamber is not more than the focal length of the confocal microscope, and the reaction between the probe and the target nucleic acid can be observed with the confocal microscope. The detection of the signal from the label of the target nucleic acid can be confirmed by visual observation, but it is possible to detect the signal by a detection means to be described later, and by further connecting the analysis means to automatically analyze the probe position, the high throughput can be achieved. It can respond to inspection.

  As shown in FIG. 2, the microarray and the hybridization chamber are installed on a stage (d) capable of temperature control, and the hybridization chamber has a solution introduction tube (f) and a discharge in the upper right and lower left portions. Tube (e) is attached. Through these tubes, a target nucleic acid solution and a cleaning solution are introduced into and discharged from the chamber.

  The confocal microscope is adjusted so that the surface of the microarray is in focus. When such adjustment is performed, the fluorescence from only the target nucleic acid that forms a hybrid with the probe can be observed in a spot shape regardless of noise from the fluorescent substance present in the solution. Using this fluorescence intensity as a guide, the hybridization results of each probe are evaluated. In the method of the present invention, it is possible to detect only the signal from the label derived from the target nucleic acid that forms a hybrid with the probe to be detected, as distinguished from the label of the target nucleic acid that does not form a hybrid with the probe to be detected. it can. Therefore, an advantage of the present invention is that the probe position can be detected even in the presence of a solution containing the target nucleic acid.

  A marker probe that emits fluorescence even if it does not form a hybrid with the target nucleic acid on the microarray is prepared. Here, the marker probe is a fluorescent dye having a thiol as a functional group, and details are described in JP-A-7-27768. Using the fluorescence of these marker probes as a guide, the focus of the microscope is adjusted and measurement is started.

  Probes immobilized on the microarray used in the present invention are appropriately selected according to the purpose of use, but the probes of the present invention can be DNA, RNA, cDNA, PNA, or other nucleic acids. It is preferable to carry out suitably.

  As long as these nucleic acid probes have a base length of a probe generally used in a microarray, the method of the present invention can be applied without any problem. In particular, the method of the present invention can be suitably used for a nucleic acid probe that is an oligonucleotide of 10 to several tens of bases that can suitably design a target nucleic acid having the same length.

  An immobilized probe nucleic acid in which a probe nucleic acid is immobilized on a carrier can be produced by an inkjet method or the like, and can be suitably produced using, for example, the method described in JP-A-11-187900.

  The composition of the hybridization solution containing the nucleic acid may be any composition that can form a hybrid, and a hybridization solution having a composition usually used in this field can be used.

  The fluorescently labeled target nucleic acid used in the present invention contains a sequence complementary to the probe whose position is to be confirmed, and forms a hybrid. Even if it forms a hybrid with other probes, it can be detected. Any level that can be distinguished from the target probe may be used. As the target nucleic acid, for example, a nucleic acid having the same length as the probe and a completely complementary sequence can be preferably used.

  When the fluorescently labeled target nucleic acid is an oligonucleotide, it can be prepared by chemical synthesis by 1-base extension, and the resulting oligonucleotide can be purified to a high purity close to 100% using a technique such as liquid chromatography. It can be purified by.

  On the other hand, when using a fluorescently labeled target nucleic acid obtained by adding a labeled molecule to a biologically derived nucleic acid, a fluorescently labeled base is used as a substrate raw material, or PCR is performed using a fluorescently labeled primer. It can be prepared by a method such as

  In this case, if the base sequence of the target nucleic acid is long and there is a sequence that is complementary to a probe other than the probe whose position is to be confirmed, Is considered to emit fluorescence. Such a target nucleic acid can be suitably used for the purpose of confirming whether or not the arrangement of a plurality of probes related to the corresponding target nucleic acid derived from a living body has reproducibility in a form including the distribution of fluorescence luminance.

  The nucleic acid microarray probe position information acquisition system according to the present invention performs a hybridization reaction between a probe on a microarray and a target nucleic acid, and observes the formed hybrid with a confocal microscope to confirm the position of the probe. It is an apparatus system for. This apparatus system has the following components in addition to the confocal microscope. First, it has a hybridization chamber having an opening surface that can be in close contact with the microarray substrate, including a probe fixing region on the nucleic acid microarray, and at least two communication ports. Furthermore, this apparatus system includes means for introducing a liquid such as a reagent for hybridization reaction, a solution containing a target nucleic acid, a buffer for washing, and the like from a communication port of the chamber and a means for discharging the liquid from the chamber. Have. Further, it has a temperature control means that has a heat source and can supply heat while controlling the temperature in the hybridization chamber. When detecting the fluorescent label, this apparatus system includes a means for generating and irradiating light for exciting the fluorescent label such as a laser, and a detecting means for detecting fluorescence from the fluorescent label such as a CCD camera. You may have. Furthermore, you may have an analyzer which receives the signal from the fluorescent label detected by the detection means, and analyzes probe position information.

(Example 1)
The detailed example of each process in an Example is shown below. However, the following examples are merely examples, and the present invention is not limited to the following specific methods, reagents, and products.

(Configuration of microarray and oligo DNA used)
A microarray prepared by the method according to the above-mentioned JP-A-11-187900 is prepared. The probe arrangement on the microarray is shown in FIG. 18-mer DNA having a thiol group at the 3 ′ end (5 ′ ACTGGCCGTCGGTTTTACA 3 ′, probe B; SEQ ID NO: 1) and 18-mer DNA also having a thiol group at the 3 ′ end (5′TGAGTCTCTAGCTAAGTCAG-SH, probe D A marker probe in which a thiol is added to a fluorescent dye (tetramethylrhodamine) is arranged and drawn as shown in FIG. A sequence complementary to probe B (5 ′ TGTAAAACGACGGCGCGT-Rhodamine; SEQ ID NO: 3) and not complementary to probe D, a target A having tetramethylrhodamine at the 3 ′ end, a sequence complementary to probe D (5 A target C (SEQ ID NO: 5) having the same sequence as that of target C and target C having tetramethylrhodamine at the 3 ′ end in “CTGACTTAGCTAGACTCA Rhodamin; SEQ ID NO: 4) was prepared. These synthetic oligo DNAs were purchased from Bex Corporation.

(blocking)
The following blocking reaction is performed before the hybridization reaction. The blocking of the microarray is performed to prevent the nucleic acid molecules from adsorbing to portions other than the probe of the microarray. Generally, it is performed immediately before the hybridization reaction.

  BSA (bovine serum albumin Fraction V: manufactured by Sigma) is dissolved in 100 mM NaCl / 10 mM phosphate buffer to 1 wt%, and the DNA microarray is immersed in this solution at room temperature for 2 hours. After blocking, wash with 0.1xSSC solution (trisodium citrate and NaCl) containing 0.1wt% SDS (sodium dodecyl sulfate) and SSC solution without SDS, rinse with ultrapure water, spin Drained with water.

(Hybridization reaction and fluorescence observation)
The drained microarray is placed on the temperature-adjustable table d in FIG. 1, and the hybridization chamber (b) is installed thereon. The hybridization chamber is firmly fixed in such a way that the microarray c is sandwiched between the stage d with temperature adjustment function and the hybridization chamber b so that there is no liquid leakage.

  Next, a hybridization solution (1M NaCl / 50 mM phosphate buffer (pH 7.0), 0.1% SDS) containing no target nucleic acid is introduced from the introduction tube f in the figure, and the temperature is raised to 75 ° C. to increase the temperature. Familiarize the probe in the hybridization chamber. After so in the solution for about 3 minutes, the temperature is slowly lowered to 50 ° C. When the temperature drops and stabilizes, set up the confocal microscope. The confocal microscope is an LSM 510 of Carl Zeiss, which uses a helium neon laser wavelength of 543 nm to observe the dye rhodamine used. Since the spot of the marker dye is fixed so as to surround the probe on the substrate, the microscope is adjusted so that the marker dye is focused in order to focus on the surface on which the probe is fixed.

  The hybridization solution containing the fluorescently labeled target nucleic acid (target A) is introduced from the introduction tube f again. After the hybridization chamber is sufficiently filled with the hybridization solution containing the target nucleic acid, the temperature is gradually decreased. The optimum temperature for the hybridization reaction depends on the base sequence and base length of the probe on the microarray and the target nucleic acid to be introduced, but it is several times higher than the Tm (melting temperature) in the liquid layer system between these complementary strands. The temperature is lowered to about 10 to several degrees. Here, a hybridization reaction was performed at 45 ° C. for 2 hours using a sequence having a length of 18 bases.

  As the temperature is lowered, it can be seen that the spot at the probe B position starts to fluoresce in about 30 minutes. By confirming at which position the probe shines, it is possible to confirm whether the predetermined array is fixed at the predetermined position.

  After confirmation of the probe, the temperature in the hybridization chamber was raised to 90 ° C. and held for 1 minute, and then a washing solution (0.1 wt% SDS (sodium dodecyl sulfate) was added from the introduction tube f in the figure. Introduce 2x SSC solution (trisodium citrate and NaCl) and flow for 20 seconds. Thereafter, with the chamber temperature set at 70 ° C., the washing solution is slowly allowed to flow for 2 minutes to remove the fluorescently labeled target nucleic acid remaining in the hybridization chamber. At this time, the speed of the flow liquid is controlled so that the temperature in the chamber does not drop below 68 ° C. by the flow liquid.

  After the washing is finished, the temperature is lowered to the temperature (45 ° C.) at which the hybridization reaction was carried out and waits for about 10 minutes. This time, it is confirmed whether the corresponding probe does not emit fluorescence. When fluorescence is observed, washing is not sufficient, so the temperature is raised again and the washing solution is flowed.

  After confirming sufficient washing, the temperature of the hybridization chamber was again maintained at 50 ° C., and another target nucleic acid (target D) was introduced from the introduction tube (f). When the temperature was slowly lowered to 45 ° C. and observed, fluorescence from the spot portion corresponding to the probe C was observed.

(Example 2)
As in Example 1, a confocal microscope is placed on the hybridization chamber, the focal plane is adjusted using a marker probe, and then the target nucleic acid is introduced from the introduction tube f. Previously, only one type of target nucleic acid labeled with one type of fluorescent dye was introduced. Here, two kinds (fitc, rhodamine) of dyes were used, and a hybridization solution in which two kinds of target nucleic acids (target A, target E) labeled with the respective dyes were mixed was introduced.

  Similarly, when the temperature of the hybridization chamber was decreased, the fluorescence of two corresponding probes could be observed. However, in the confocal microscope, a helium neon laser having a wavelength of 543 nm and an argon laser having a wavelength of 477 nm were used corresponding to two types of dyes, and both were separately observed by a filter.

(Example 3)
As shown in FIG. 4, a substrate on which 20 types of probes were printed was prepared. A target nucleic acid having a sequence complementary to each probe, which is labeled with rhodamine for those complementary to probes 11 to 20 and labeled with fitc for those complementary to probes 21 to 30, is prepared. The target nucleic acid 11 to the target nucleic acid 30 are designated for the corresponding probes.

  As in Example 2, helium neon laser (543 nm) and argon laser (477 nm) were set, and the target nucleic acid 11 and target nucleic acid 21 were introduced into the hybridization chamber maintained at 50 ° C. from the introduction tube (f). The temperature was slowly lowered. After maintaining at 45 ° C. for about 15 minutes, the fluorescence of rhodamine and fitc was confirmed on probe 11 and probe 21, respectively.

  After the washing liquid flow at 75 ° C. for 5 minutes, the target nucleic acid 12 and the target nucleic acid 22 were introduced. And it confirmed that the position of the probe 12 and the probe 22 emitted fluorescence.

  The same process was repeated 10 times, and all 20 types of probes could be confirmed in order.

It is a general view which shows the implementation condition of this invention. It is an enlarged view of a hybridization chamber part. FIG. 3 is a probe layout diagram of a microarray used in Examples 1 and 2. FIG. 6 is a probe layout diagram of a microarray used in Example 3.

Explanation of symbols

a Confocal microscope b Hybridization chamber c Microarray d Temperature-controlled base e Drain pipe f Inlet pipe

Claims (3)

A method for obtaining positional information of probes immobilized on a nucleic acid microarray,
(I) introducing a solution containing at least one type of target nucleic acid having a sequence complementary to the probe into a probe fixing region to which a plurality of types of probes are fixed on the microarray; Performing a hybridization reaction with the probe;
(Ii) After the reaction, in the presence of the solution, the surface on which the probe fixing region is present is observed with a confocal microscope, and the position of the probe is confirmed from the signal from the detected target nucleic acid label. And a process of
(Iii) removing the target nucleic acid introduced in step (i) with a washing buffer;
A method for acquiring probe position information, wherein the steps (i) to (iii) are repeated for the same microarray.   In the step (i), when simultaneously introducing a solution containing a plurality of types of target nucleic acids, each of the plurality of types of target nucleic acids is labeled with a different type of label. The method described.   The method for obtaining probe position information according to claim 1, wherein, in the step (iii), the washing buffer is introduced at a temperature at which the hybrid formed in the step (i) is dissociated. .

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