JP4101810B2 - Microarray regeneration method and microarray regeneration reagent kit - Google Patents

Description

  The present invention relates to a microarray regeneration method and a regeneration reagent kit used in the field of biotechnology such as gene diagnosis, DNA sequence analysis, or gene polymorphism analysis, particularly in the field of genetic testing.

A DNA chip / microarray (hereinafter referred to as a microarray) is a DNA in which several hundred to tens of thousands of spots of DNA serving as a probe are arranged on a substrate such as a glass slide or silicon. According to the microarray, it is possible to provide a technique capable of collectively and collectively processing operations that require hundreds to tens of thousands of experiments in the conventional method. Therefore, microarrays have become an important technology especially in fields requiring high throughput such as searching for disease-related genes, such as life science fields such as pharmaceuticals.
In major microarrays currently in use, a solution sample containing nucleic acids amplified by the PCR method or the like is flowed on the microarray, and hybridization between the probe on the microarray and the amplified nucleic acid is detected, thereby allowing genetic diagnosis, gene We perform polymorphism analysis and DNA sequence analysis.
On the other hand, a microarray using the extension of a probe on a solid phase can simultaneously perform hybridization with the probe and amplify the nucleic acid, so that real-time detection is possible.
However, since these microarrays perform nucleic acid amplification from the probe, the length of the probe changes before and after use and cannot be reused. Moreover, these microarrays are expensive despite being used up once. In order to make a low-cost microarray, it is useful to use it repeatedly. Especially, the ability to repeatedly use a microarray integrated with nucleic acid amplification and detection is very useful from the viewpoint of greatly reducing the detection time. I can say that. However, in the microarray integrated with nucleic acid amplification / detection, the immobilized probe is extended, so that the process of returning the extended probe to its original length when used next time is from the viewpoint of control of the hybridization temperature. It is essential.
In order to solve this problem, there is a method of using a peptide nucleic acid (PNA) as a probe. Peptide nucleic acid is a substance with a peptide backbone in which the sugar-phosphate part of the DNA backbone structure is replaced with (2-aminoethyl) glycine, and has a three-dimensional structure and chemical properties similar to nucleic acid. It can be a substitute for nucleic acid. PNA is chemically synthesized by applying a solid phase synthesis method for peptides, but can be used as a probe to form a strong complementary chain bond with DNA. PNA molecules are neutral substances with no chiral centers and excellent solubility, and are resistant to nucleolytic enzymes. Therefore, in a microarray using PNA as a probe, after the extension reaction from the probe is performed using dNTP as a substrate, only the extended region (dNTP portion) is cleaved and removed by flowing a nucleolytic enzyme on the microarray. It is possible to hold the PNA as a probe on the substrate.
However, in a microarray using PNA as a probe, it is known that aggregation occurs when the purine content (adenine and guanine) of the PNA probe exceeds 60%. Therefore, in a microarray using PNA as a probe, the arrangement of the probe is limited, and there is a problem that the range that can be detected is limited.
On the other hand, in a microarray to which a DNA probe is immobilized, a method of excising an extended strand using a restriction enzyme has been devised. However, in this microarray, it is necessary to select an appropriate restriction enzyme for each probe, and the extended strand cannot be excised by a uniform treatment. Therefore, this microarray has a problem that the cost cannot be reduced.
There is also a method in which in a microarray to which a DNA probe is immobilized, the substrate is extended using dUTP instead of dTTP, and the extended strand containing dUTP is excised using uracil N glycosylase. However, this microarray has a problem that the extended chain cannot be cleaved at a desired site.
Therefore, an object of the present invention is to provide a microarray regeneration method and a microarray regeneration reagent kit that can excise an extended chain amplified by an extension reaction from a DNA probe at a low cost.

The microarray regeneration method according to the present invention that achieves the above-described object is the method of carrying out the extension reaction from the 3 ′ end of the DNA probe on the microarray having the DNA probe having the 5 ′ end immobilized on the substrate. A step of reacting an oligonucleotide having a thymidine complementary to the extended strand amplified by EDTA and bound with EDTA (ethylenediaminetetraacetic acid), a divalent metal, and a Cleland reagent, the divalent metal And cleaving the hybridized oligonucleotide and the extended strand together with the Cleland reagent.
The microarray regeneration reagent kit according to the present invention is a sequence complementary to an extended strand amplified by an extension reaction from the 3 ′ end of the DNA probe of a microarray having a DNA probe having a 5 ′ end immobilized on a substrate. An oligonucleotide having thymidine to which EDTA is bound, a divalent metal, and a Cleland reagent.
According to the present invention, the divalent metal and the Cleland reagent are allowed to act in a state where the oligonucleotide is hybridized to the extended strand amplified by the extension reaction from the 3 ′ end of the DNA probe. In the presence of a divalent metal, the Cleland reagent cleaves double-stranded and / or single-stranded DNA containing thymidine bound to EDTA in the vicinity of thymidine bound to the EDTA. Thereby, the extended strand can be excised from the DNA probe. The microarray from which the extended strand has been excised can be used again.
Hereinafter, the above and other novel features and effects of the present invention will be described in detail with reference to the drawings. However, the technical scope of the present invention is not limited to the following description. In the drawings used for description, the same functional parts are denoted by the same reference numerals.

FIG. 1 is a schematic diagram showing a solid phase amplification type microarray. FIG. 2 is a schematic diagram showing a state of nucleic acid probe extension of the microarray.
FIG. 3 is a schematic diagram showing a state of nucleic acid probe cleavage of the microarray.
FIG. 4 is a schematic diagram showing a state of nucleic acid amplification of the microarray.
FIG. 5 is a schematic diagram showing a state of hybridization between the DNA probe of the microarray and the oligonucleotide.
FIG. 6 is a schematic diagram showing a state of nucleic acid probe regeneration of the microarray.

As shown in FIG. 1, the microarray applied to the present invention includes a substrate 1 and a plurality of DNA probes 2 immobilized on the substrate 1.
The board | substrate 1 is not specifically limited, For example, it consists of a glass slide, a metal, nylon, or a nitrocellulose membrane. In addition, the substrate 1 may be one in which a part or all of the surface is subjected to surface treatment, or a film made of aminosilane may be formed.
The DNA probe is composed of nucleic acid fragments, and is formed by immobilizing the nucleic acid fragments on one main surface of the substrate 1 at a predetermined density. Here, immobilization means not only a covalent bond but also any bonding mode such as an ionic bond, and also includes both a bond via a linker and a bond not via a linker. The nucleic acid fragment to be immobilized is not particularly limited, and examples thereof include a DNA fragment extracted from a specimen and prepared by a process such as amplification and purification, and a synthesized oligo DNA. In addition, a so-called spotter (arrayer) can be used for immobilizing nucleic acid fragments.
Further, in the DNA probe shown in FIG. 1, the 5 ′ end side of the nucleic acid fragment is immobilized on the substrate 1, and the 3 ′ end side has regions 3 and 4 capable of hybridizing with the DNA to be detected. . For example, the region 3 and the region 4 can be designed so that they can hybridize with different DNAs. Specifically, when detecting the SNP, the region 3 corresponding to one SNP and the region 4 corresponding to the other SNP are designed and immobilized at different sites.
When using the microarray configured as described above, first, a DNA 6 hybridized to the immobilized DNA probe 2 is used as a template, and a nucleic acid is subjected to an extension reaction from the 3 ′ end of the DNA probe. At this time, by performing an extension reaction with a so-called strand displacement enzyme, it is possible to sequentially perform an extension reaction using the DNA hybridized under normal temperature conditions as a template, and amplify the desired DNA (FIGS. 1 to 4). ).
Specifically, first, as shown in FIG. 1, after placing the microarray on the flow cell, the reaction reagent is allowed to act on the microarray from the reagent injection port 7. Here, the reaction reagent is a sample containing DNA 6 to be detected, a DNA elongation enzyme (for example, a strand displacement enzyme), dNTP, and a DNA cleavage enzyme. Making the reaction reagent act on the microarray means bringing the reaction reagent into contact with the surface of the substrate 1 on which the DNA probe 2 is immobilized. By bringing the reaction reagent into contact with the surface of the substrate 1, the DNA 6 and the DNA probe 2 contained in the reaction reagent are specifically hybridized as shown in FIG.
At this time, when the regions 3 and 4 are designed to detect a predetermined SNP, one or both of the region 3 and the region 4 are corresponding to the SNP in the detection target DNA 6 included in the sample. Thus, the DNA to be detected is hybridized.
Further, in this state, as shown in FIG. 2, the DNA extender contained in the reaction reagent uses DNA 6 hybridized with the DNA probe 2 as a template, and uses dNTP as a substrate from the 3 ′ end of the DNA probe 2. Extend the nucleic acid. For example, commercially available Taq polymerase, Bst polymerase, Bac polymerase, etc. can be used as the DNA elongation enzyme.
As shown in FIG. 3, the DNA-cleaving enzyme is an enzyme that can nick a DNA strand extended by a DNA extending enzyme. For example, as a DNA cleaving enzyme, a restriction enzyme that can recognize a specific sequence and can nick the extended strand side can be used. As this restriction enzyme, commercially available EcoRI polymerase, HindIII polymerase and the like can be used.
In this example, by using a strand displacement type DNA extender as the DNA extender, as shown in FIG. 4, the DNA extension reaction is started again from the nick formed by the DNA cleaving enzyme, and it has already been formed. A new extension strand can be synthesized while peeling the complementary strand from the template DNA.
Thus, by using a microarray, an extension reaction can be performed with a so-called strand displacement enzyme, and the DNA can be sequentially extended as shown in FIGS. 3 and 4 using a DNA hybridized at room temperature as a template. Can be amplified. After the above steps are completed, the reaction reagent remaining on the microarray and the DNA 6 hybridized to the DNA probe 2 are removed, and then the microarray can be regenerated as follows.
After the experiment is completed, the length of the extended nucleic acid probes 2 and 3 needs to be restored to the next experiment of the microarray in order to control the hybridization temperature (Tm). Therefore, in the present invention, the reagent for microarray regeneration is injected from the reagent injection port 7.
Here, the reagent for microarray regeneration includes an oligonucleotide having a thymidine complementary to an extended strand amplified by an extension reaction from the 3 ′ end of the DNA probe 2 and having EDTA (ethylenediaminetetraacetic acid) bound thereto, A valent metal and a Cleland reagent are included. Here, the oligonucleotide is designed to specifically hybridize with the extended strand remaining on the 3 ′ side of the DNA probe 2 and contains thymidine bound with EDTA in at least one position.
As the divalent metal, divalent iron [Fe (II)] is preferably used, but is not limited thereto, and for example, Mg ²⁺ or Mn ²⁺ may be used. Further, as the Cleland reagent, for example, dithiothreitol can be used, but not limited thereto, dithioerythritol and mercaptoethanol can be used.
When a reagent for microarray regeneration is injected from the sample injection port 7, the reagent can be brought into contact with the surface of the substrate 1 on which the DNA probe 2 is immobilized. Thereby, the oligonucleotides 10 and 11 contained in the reagent for microarray regeneration specifically hybridize to the extended strand remaining on the 3 ′ side of the DNA probe 2 as shown in FIG.
In this state, the Creeland reagent can cleave DNA on the extended strand side in the vicinity of thymidine to which EDTA is bound in the presence of a divalent metal. After the extension strand is cleaved by the Cleland reagent, heat denaturation is performed by heat treatment at 94 to 95 ° C., and then the microarray regeneration reagent containing the oligonucleotides 10 and 11 is removed from the surface of the substrate 1 of the microarray to obtain FIG. As shown, the DNA probe 2 in place of the microarray can be regenerated.
In this step, the oligonucleotide, the divalent metal, and the Cleland reagent may be allowed to act on the microarray at the same time, but first, only the oligonucleotides 10 and 11 are allowed to act on the DNA probe 2 and the oligonucleotides 10 and 11. After hybridizing, a divalent metal and a Cleland reagent may be allowed to act. Alternatively, the oligonucleotides 10 and 11 and one of the divalent metal and the Cleland reagent may be allowed to act simultaneously, and then either the other of the divalent metal and the Cleland reagent may be allowed to act.
In this example, it is preferable that the oligonucleotides 10 and 11 have a complementary sequence at a position 5 or more bases away from the 3 ′ end of the DNA probe 2 in the extended strand. According to the Cleland reagent, DNA on the extended strand side is cleaved in the vicinity of thymidine to which EDTA is bound, and therefore within 5 bases may be excised from thymidine to which the EDTA is bound. Therefore, by making the oligonucleotides 10 and 11 complementary sequences in the extended strand at a position 5 bases or more away from the 3 ′ end of the DNA probe 2, the DNA probe 2 is cut when the extended strand DNA is cleaved by the Cleland reagent. Can be prevented from being excised.
By the way, as described above, the microarray to which the present invention is applied is not limited to the one in which an extension reaction from a DNA probe is performed by a strand displacement type DNA extension enzyme. For example, a heat-resistant DNA polymerase is used and temperature control is performed. An extension reaction from a DNA probe may be performed.

Industrial applicability

  As described above in detail, the present invention can provide a microarray regeneration method and a regeneration reagent kit that can repeatedly use a microarray utilizing the extension of a probe on a solid phase. According to the present invention, it is possible to reduce costs in the field of biotechnology such as DNA sequence analysis or gene polymorphism analysis, particularly in the field of genetic testing such as diagnosis and treatment that requires detection of a target nucleic acid. it can.

Claims (9)

After performing an extension reaction from the 3 ′ end of the DNA probe on a microarray having a DNA probe having the 5 ′ end immobilized on the substrate, the sequence is complementary to the extended strand amplified by the extension reaction, and is an EDTA ( A step of reacting an oligonucleotide having thymidine bound with ethylenediaminetetraacetic acid), a divalent metal, and a Cleland reagent,
Cleaving the hybridized oligonucleotide and the extended strand together with the divalent metal and the Cleland reagent;
A microarray regeneration method comprising: The microarray regeneration method according to claim 1, further comprising a step of washing the microarray. 2. The microarray regeneration method according to claim 1, wherein the Cleland reagent is dithiothreitol, and the divalent metal is divalent iron. The oligonucleotide is a region complementary to a sequence containing 5 to 10 bases from a base that is 5 or more bases away from the 3 ′ end of the DNA probe in the extended strand and containing adenine. The microarray reproduction method according to claim 1. 2. The microarray regeneration method according to claim 1, wherein the oligonucleotide has a sequence complementary to a region not containing a polymorphism. A thymidine that is complementary to an extended strand amplified by an extension reaction from the 3 ′ end of the DNA probe of a microarray having a DNA probe immobilized on the substrate at the 5 ′ end, and to which EDTA (ethylenediaminetetraacetic acid) is bound An oligonucleotide having
Bivalent metals,
A reagent kit for microarray regeneration, comprising Cleland reagent. The microarray regeneration reagent kit according to claim 6, wherein the Cleland reagent is dithiothreitol, and the divalent metal is divalent iron. The oligonucleotide is a region complementary to a sequence containing 5 to 10 bases from a base that is 5 or more bases away from the 3 ′ end of the DNA probe in the extended strand and containing adenine. The microarray regeneration reagent kit according to claim 6. The microarray regeneration reagent kit according to claim 6, wherein the oligonucleotide has a sequence complementary to a region not containing a polymorphism.

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