JP2009089696A - Carrier for gene subtraction and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tool for enabling a gene subtraction method to be applied simply, rapidly and highly accurately to wide genes, and to provide the gene subtraction method by using the tool and applicable to the wide genes simply, rapidly and highly accurately. <P>SOLUTION: The carrier for the gene subtraction is obtained by binding a nucleic acid having a nucleotide sequence annealing with a nucleotide sequence common to a gene family targeted by the gene subtraction. The gene subtraction method uses the carrier. The gene family targeted by the gene subtraction is preferably a protein kinase gene family, and the protein kinase gene family preferably comprises a tyrosine kinase family and a serine/threonine kinase family. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel carrier for gene subtraction and a novel gene subtraction method using the same.

First, the abbreviations used in the application documents will be described.
G3PDH: glyceraldehyde 3-phosphate dehydrogenase
OA: osteoarthritis
PCR: Polymerase Chain Reaction
PK: protein kinase
RA: rheumatoid arthritis
SDS: sodium dodecyl sulfate
In the present application documents, the following abbreviations are used to indicate nucleotide sequences (base sequences).
A (or a): adenine G (or g): guanine C (or c): cytosine T (or t): thymine U (or u): uracil R (or r): guanine or adenine Y (or y): Thymine or cytosine M (or m): adenine or cytosine S (or s): guanine or cytosine W (or w): adenine or thymine N (or n): adenine or guanine or cytosine or thymine Gene subtraction method (subtraction hybrid method) ) Is a method of isolating a target gene having a difference in expression level between two different types of cells and tissues by subtraction through hybridization. For example, when mRNA of a certain target gene is expressed at a higher level in cell B than in cell A, a hybrid is formed between mRNA / cDNA / cRNA derived from cells A and B, and the hybrid is formed. By removing things other than those, genes that are expressed at a high level in cell B can be isolated.

In addition, many gene subtraction kits are commercially available. However, as a result of performing gene subtraction on a cDNA sample containing an equal amount of JAK1 (a type of tyrosine kinase) by the inventors using a commercially available kit, JAK1 originally to be removed by gene subtraction In some cases, cDNA was hardly removed and a considerable amount remained. This demonstrates the state of the art that even a well-established and widely available kit is not necessarily versatile for a wide variety of gene subtraction, and there is ample room for improvement in the gene subtraction method. It became to show that there was. In addition, since all the expressed genes are targeted by the conventional subtraction method, a large number of genes including genes with unknown functions are detected. Therefore, it is very difficult and inefficient to select a meaningful gene from the detected genes.

The present invention provides a tool for making the gene subtraction method applicable to a wide range of genes more easily, quickly, with high accuracy, and further, with respect to a simple, rapid, high accuracy, and wide range of genes using this method. It is an object of the present invention to provide a gene subtraction method that can be applied. Another object of the present invention is to increase the efficiency of gene subtraction by analyzing only genes with known and meaningful functions, that is, specific gene families.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have produced a carrier to which “a nucleic acid having a nucleotide sequence that anneals to a nucleotide sequence common to a gene family targeted for gene subtraction” is bound. By using this for gene subtraction, it was found that gene subtraction applicable to a wide range of genes was possible simply, quickly, with high accuracy, and the present invention was completed.

That is, the present invention relates to a carrier for gene subtraction (hereinafter referred to as “a nucleic acid having a nucleotide sequence that anneals to a nucleotide sequence common to a gene family targeted for gene subtraction”) "The carrier of the present invention").
This “gene family targeted for gene subtraction” is preferably the PK gene family. The “PK gene family” is preferably the tyrosine kinase family and the serine / threonine kinase family.
Moreover, when the PK gene family is selected as the “gene family targeted for gene subtraction”, the nucleotide sequence common to the family is preferably the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2.
GTGCACMGNGAYYT (SEQ ID NO: 1)
GAYGTSTGGTCCTWTGG (SEQ ID NO: 2)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
In addition, when the PK gene family is selected as the “target gene family for gene subtraction”, the nucleotide sequence annealed to the nucleotide sequence common to the family is the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3. Preferably there is.
GTGCACMGNGAYYT (SEQ ID NO: 1)
CCAWAGGACCASACRTC (SEQ ID NO: 3)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
In addition, when the PK gene family is selected as the “target gene family for gene subtraction”, the nucleotide sequence annealed to the nucleotide sequence common to the family is the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 4. Preferably there is.
GTGCACMGNGAYYT (SEQ ID NO: 1)
GCGAATTCCAWAGGACCASACRTC (SEQ ID NO: 4)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
The “carrier” is preferably a magnetic bead. The binding mode between the nucleic acid and the carrier is preferably a biotin-avidin bond.
The “nucleic acid” is preferably DNA.

  The present invention also provides a gene subtraction method (hereinafter referred to as “the method of the present invention”) characterized by using the carrier of the present invention. The method of the present invention includes a step of bringing a “nucleic acid bound to the carrier of the present invention” and a “nucleic acid fraction containing the nucleic acid to be detected” into contact for hybridization, and then removing the carrier. It is preferable.

  According to the present invention, there is provided a tool for making the gene subtraction method simpler, faster, more accurate, and applicable to a wide range of genes. Since a gene subtraction method applicable to is provided, it is extremely useful.

Hereinafter, the present invention will be described in detail by embodiments of the invention.
<1> Carrier of the present invention The carrier of the present invention is a gene subtraction characterized in that “nucleic acid holding a nucleotide sequence that anneals to a“ nucleotide sequence common to a gene family targeted for gene subtraction ”” is bound. Carrier.

  Here, as the “gene family targeted for gene subtraction”, a desired gene subtraction target (target) may be selected from various gene families. For example, when gene subtraction is desired for the PK gene family, the PK gene family is the gene family targeted for gene subtraction.

In the present invention, the PK gene family is preferably selected as the “gene family targeted for gene subtraction”. The “PK gene family” is preferably the tyrosine kinase family and the serine / threonine kinase family.
The “common nucleotide sequence” for the gene family targeted for gene subtraction is not particularly limited as long as it is a nucleotide sequence that is commonly present in the family. Here, “commonly present” does not necessarily have to exist in all genes belonging to the gene family, and may be any gene that exists in many genes belonging to the gene family. Further, the “common nucleotide sequence” itself does not necessarily need to be completely matched between the genes, and there may be slight nucleotide differences.
In the gene family targeted for gene subtraction, the specific nucleotide sequence to be used as the “common nucleotide sequence” can be appropriately determined by those skilled in the art depending on the purpose and accuracy of subtraction. For example, if you want to perform gene subtraction for a certain gene family to some extent, the “nucleotide sequence common to the gene family” belongs to the gene family instead of the nucleotide sequences that exist in all the genes belonging to the gene family. Nucleotide sequences that exist in many genes may be used. Conversely, for a gene family, if you want to perform gene subtraction with a strict focus on specific nucleotide sequences present in all genes belonging to the gene family, What is necessary is just to use the said specific nucleotide sequence which exists in all the genes which belong to a gene family.
When the PK gene family is selected as the “gene family targeted for gene subtraction”, it is preferable to employ the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 as the nucleotide sequence common to the family. .
GTGCACMGNGAYYT (SEQ ID NO: 1)
GAYGTSTGGTCCTWTGG (SEQ ID NO: 2)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
Among them, it is preferable to adopt both nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 as nucleotide sequences common to the family.
In addition, “the nucleotide sequence that is annealed to the“ nucleotide sequence common to the gene family targeted for gene subtraction ”” refers to the “nucleotide sequence common to the gene family targeted for gene subtraction” described above, There is no particular limitation as long as the nucleotide sequence has the property of annealing. Here, the condition of “annealing” is that when the temperature is lowered from the temperature condition in which the hydrogen bonds between the nucleic acid strands to be complementarily formed are broken, the hydrogen bonds are reconfigured between the nucleic acid strands. As long as the conditions are such that they are recombined, there is no particular limitation. Specifically, the conditions in which recombination is formed between the nucleic acid strands in the cooling step in PCR can be mentioned. More specifically, it is preferable that recombination between the nucleic acid strands is formed at 48 ° C. (˜70 ° C.) for 1 minute using the buffer attached to ExTaq polymerase (TaKaRa: RR001A). .
When the PK gene family is selected as the “target gene family for gene subtraction”, the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 3 is used as the nucleotide sequence that anneals to the nucleotide sequence common to the family. It is preferable to adopt.
GTGCACMGNGAYYT (SEQ ID NO: 1)
CCAWAGGACCASACRTC (SEQ ID NO: 3)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
Among these, it is preferable to employ the nucleotide sequences of both SEQ ID NO: 1 and SEQ ID NO: 3.
In addition, when a PK gene family is selected as the “target gene family for gene subtraction”, the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 4 is used as a nucleotide sequence that anneals to a nucleotide sequence common to the family. Is preferably adopted.
GTGCACMGNGAYYT (SEQ ID NO: 1)
GCGAATTCCAWAGGACCASACRTC (SEQ ID NO: 4)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
Among these, it is preferable to employ the nucleotide sequences of both SEQ ID NO: 1 and SEQ ID NO: 4.

  Based on such a nucleic acid consisting of “a nucleotide sequence that is annealed to a“ nucleotide sequence common to a gene family targeted for gene subtraction ””, a nucleic acid that retains the sequence to be bound to a carrier can be produced. it can. In this application document, a nucleic acid that “holds” a nucleotide sequence is intended to include not only a nucleic acid consisting of the nucleotide sequence but also a nucleic acid partially containing the nucleotide sequence.

As described above, in the gene subtraction method, when mRNA of a certain target gene is expressed at a higher level in the cell B than in the cell A, a hybrid between mRNA / cDNA / cRNA derived from the cells A and B, etc. Genes that are expressed at a high level in cell B can be isolated by removing those other than those that are hybridized. In this application document, the nucleic acid derived from cell A (control sample) that forms a hybrid in this case is referred to as “driver”, and the nucleic acid derived from cell B (target sample) is referred to as “tester”. The “driver” in the present invention is a “nucleic acid having a nucleotide sequence that anneals to a nucleotide sequence common to a gene family targeted for gene subtraction” that is expressed in the cell A. As the “tester”, “nucleic acid having a nucleotide sequence that anneals to“ a nucleotide sequence common to a gene family targeted for gene subtraction ”” that is expressed in the cell B is used.
The “nucleic acid having a nucleotide sequence that anneals to a“ nucleotide sequence common to the gene family targeted for gene subtraction ”” to be bound to the carrier is preferably a nucleic acid used as a “driver”. Such a nucleic acid is a nucleic acid comprising the above-mentioned “nucleotide sequence that is annealed to a“ nucleotide sequence common to the gene family targeted for gene subtraction ”” as a primer, and a control cell / tissue or the like in the gene subtraction (as described above). It can be produced and amplified by PCR using a nucleic acid (such as mRNA) present in “cell A”) as a template.
For example, when the PK gene family is selected as the “target gene family for gene subtraction” and the synovial membrane of OA patients is used as a control for gene subtraction (“cell A” as described above), SEQ ID NO: 1 and The nucleic acid to be bound to the carrier can be produced / amplified by PCR using the nucleic acid represented by SEQ ID NO: 3 as a primer and the nucleic acid extracted from the synovial membrane of an OA patient as a template. In place of SEQ ID NO: 3, the nucleic acid represented by SEQ ID NO: 4 may be used.
GTGCACMGNGAYYT (SEQ ID NO: 1)
CCAWAGGACCASACRTC (SEQ ID NO: 3)
GCGAATTCCAWAGGACCASACRTC (SEQ ID NO: 4)
(T in the sequence number may be U. T in Y, W, and N may also be U.)
As described above, the “nucleic acid having a nucleotide sequence that anneals to the“ nucleotide sequence common to the gene family targeted for gene subtraction ”” to be bound to the carrier of the present invention refers to the gene family targeted for gene subtraction and the control. Depending on the cell, tissue, etc. ("driver" used), etc., the person skilled in the art can appropriately set and manufacture according to these.

  The carrier of the present invention is characterized in that such a “nucleic acid holding a nucleotide sequence that anneals to a“ nucleotide sequence common to a gene family targeted for gene subtraction ”” is bound to the carrier.

The “carrier” is not particularly limited as long as it can bind a nucleic acid and is insoluble in water, a reaction solution or the like. The shape is not particularly limited, and examples thereof include beads, plates (for example, microplate wells), tubes, membranes, gels, and the like. Of these, the shape of beads is preferred.
The material of the carrier is not particularly limited, and examples thereof include magnetic materials, polystyrene, polypropylene, polyvinyl chloride, nitrocellulose, nylon, polyacrylamide, polyallomer, polyethylene, glass, agarose, and the like.

  Among these, magnetic beads are preferred because removal of the driver after hybridization can be performed simply, quickly and reliably. That is, when magnetic beads are used as a carrier, the beads (the nucleic acid bound to the beads and the nucleic acid hybridized to the beads) can be easily, rapidly and reliably removed by a magnet after hybridization.

Further, the binding mode between the nucleic acid and the carrier is not particularly limited as long as it has a strength higher than the strength at which the both do not dissociate in water or a reaction solution. In addition, the nucleic acid and the carrier may be directly bound together, or both may be bound via another substance.
Examples of the bond between the two include a covalent bond formed by two atoms sharing some electrons, an affinity bond formed by the affinity between two molecules, and the like. Examples of the affinity bond include biotin-avidin bond (bond between biotin and avidin) and the like. The term “avidin” in the present application document naturally includes streptavidin.

  When a biotin-avidin bond is adopted as the binding mode between the two, for example, biotin is bound to the nucleic acid to be bound to the carrier by a known method, and avidin is bound to the carrier (for example, magnetic beads). The carrier of the present invention in which the nucleic acid and the carrier are bound by a biotin-avidin bond can be obtained by bringing them into contact with each other and affinity binding. In addition, carriers (eg, magnetic beads) to which avidin and the like are bound are commercially available, and these can also be used.

  In addition, you may form the coupling | bonding of the nucleic acid and carrier in this invention support | carrier in the process of gene subtraction. For example, after the driver and the tester are hybridized, the driver may be bound to the carrier. For example, the carrier of the present invention can be produced by previously binding biotin to a driver, hybridizing it with a tester, and then binding biotin bound to the driver to avidin bound to the carrier. May be.

  The carrier of the present invention is provided by using a carrier to which a nucleic acid as described above is bound as a carrier for gene subtraction.

  The nucleotide sequences and nucleic acids shown above may be DNA or RNA, and can be appropriately selected by those skilled in the art depending on the purpose and method of gene subtraction. For example, when RNA is used as a nucleotide sequence or nucleic acid, thymine (T) may be changed to uracil (U), and when mRNA is used, the nucleotide sequence is designed in consideration of transcription from DNA. do it.

The carrier of the present invention can be used as it is in the “method of the present invention” described later. Refer to the description of the “method of the present invention” to be described later.

<2> Method of the Present Invention The method of the present invention is a gene subtraction method using the carrier of the present invention. The method of the present invention is not particularly limited as long as it includes at least the step of using the carrier of the present invention.
As described above, the carrier of the present invention is characterized in that “a nucleic acid having a nucleotide sequence that anneals to a nucleotide sequence common to a gene family targeted for gene subtraction” is bound. And as above-mentioned, it is preferable that the said nucleic acid is a nucleic acid used as a driver. Therefore, the method of the present invention can be carried out by using the carrier of the present invention instead of the driver in the gene subtraction method.

  Specifically, in the present invention, a “nucleic acid bound to the carrier of the present invention” and a “nucleic acid fraction containing a nucleic acid to be detected (tester)” are brought into contact with each other for hybridization, Preferably, the method includes a step of removing the carrier.

The hybridization reaction conditions here are preferably stringent conditions that do not cause non-specific hybridization. As such conditions, the final concentration is 0.5 to 10xSSC (preferably 1 to 5xSSC, more preferably 3xSSC), 0.5 to 5xDenhardt solution (preferably 1 to 4xDenhardt solution, more preferably 2.5xDenhardt solution).
And in a solution containing 0.01 to 0.1% SDS (preferably 0.02 to 0.08% SDS, more preferably 0.05% SDS) at 55 ° C to 75 ° C (preferably 60 ° C to 70 ° C, more preferably 68 ° C). Incubation conditions are exemplified. Examples of the incubation time include 6 hours to 24 hours (preferably 10 hours to 20 hours, more preferably 15 hours).
Here, the “nucleic acid bound to the carrier of the present invention” is as described above. The “nucleic acid fraction containing the nucleic acid to be detected” is a “tester” and can be appropriately selected and manufactured by those skilled in the art according to the purpose of gene subtraction.
For example, when gene subtraction is performed on the PK gene family whose expression level is higher in the joint synovium of the RA patient than in the synovium of the OA patient, the PK gene extracted from the joint synovium of the OA patient is amplified. Using the carrier of the present invention in which a nucleic acid group (driver) is bound to a carrier, the nucleic acid (driver) bound to the carrier and the nucleic acid fraction (tester) relating to the PK gene extracted and amplified from the RA synovial membrane And the carrier (the nucleic acid bound to the carrier and the nucleic acid hybridized to the carrier) may be removed.
In particular, when magnetic beads are used as the “carrier” in the carrier of the present invention, the carrier can be removed simply, quickly and reliably using a magnet.
For example, the method which consists of the following steps can be illustrated.
(1) RNA is extracted from the synovial membrane of human RA patients and human OA patients.
(2) Synthesize cDNA using the extracted RNA as a template.
(3) Using a cDNA derived from RA patient as a template, PCR is performed using primers specific to the PK gene family, and a fragment of the PK gene family is specifically amplified. The DNA thus obtained is referred to as “tester DNA”.
(4) PCR is performed using a cDNA derived from OA patient as a template, a primer with biotin added to the 5 ′ end of a sequence specific to the PK gene family, and a fragment of the PK gene family modified with biotin at both ends. Amplifies specifically. The DNA obtained from this is called “driver DNA”.
(5) Tester DNA and driver DNA are mixed and denatured by heating in an aqueous solution (preferably 80 ° C. to 100 ° C., more preferably 90 ° C. to 100 ° C., particularly preferably about 100 ° C.). Then, final concentrations of 0.5 to 10xSSC (preferably 1 to 5xSSC, more preferably 3xSSC), 0.5 to 5xDenhardt solution (preferably 1 to 4xDenhardt solution, more preferably 2.5xDenhardt solution) and 0.01 to 0.1% SDS (preferably 0.02) In a solution containing ˜0.08% SDS, more preferably 0.05% SDS, at 55 ° C. to 75 ° C. (preferably 60 ° C. to 70 ° C., more preferably 68 ° C.) for 6 hours to 24 hours (preferably 10 hours to Incubation (20 hours, more preferably 15 hours) causes a double strand to form between the tester DNA and the driver DNA.
(6) Magnetic beads are added to this reaction solution, and biotin in the driver DNA is bound to streptavidin that is bound to the magnetic beads.
(7) The complex of driver DNA and magnetic beads is removed using a magnet. As a result, not only the driver DNA but also the gene fragment that is common to the tester DNA and the driver DNA is removed. As a result, DNA whose expression is increased in RA patients compared to OA patients remains in the solution. This DNA is amplified by PCR and used for various analyses. In order to further increase the sensitivity and accuracy of gene subtraction, the above steps may be further repeated for the solution after removing the magnetic beads (carrier).
The same applies when targeting other cells / tissues or other gene families.

According to the method of the present invention, a gene subtraction method applicable to a wide range of genes can be performed simply, rapidly, with high accuracy.

Hereinafter, the present invention will be described more specifically with reference to examples.
<Example 1> Gene subtraction using the carrier of the present invention (1) Knee joint synovium was removed during surgery of human RA patients (6 cases) and human OA patients (3 cases), and ISOGEN (NIPPON GENE RNA was extracted by the method.
(2) Using the extracted RNA as a template, cDNA was synthesized using SuperSript III First-Strand Synthesis System (Invitrogen). The operation method was performed according to the manual of the product.
(3) PCR (reaction condition: ExTaq polymerase (TaKaRa: RR001A) using primers derived from RA patients as a template and using primers specific to the PK gene family (TAGAACTAGTGGATCCGTGCACMGNGAYYT (SEQ ID NO: 5) and GGTCGACGGTATCGATCCAWAGGACCASACRTC (SEQ ID NO: 6)) ) And the attached buffer, 94 ℃ (3 minutes)-[94 ℃ (1 minute)-48 ℃ (1 minute) -72 ℃ (1 minute)] x 30 times-72 ℃ (5 minutes)) And a fragment of the PK gene family was specifically amplified. Hereinafter, the DNA obtained by this amplification is referred to as “tester DNA”.
(4) PCR using a primer (GTGCACMGNGAYYT (SEQ ID NO: 1) and GCGAATTCCAWAGGACCASACRTC (SEQ ID NO: 4)) with biotin added to the 5 ′ end of a sequence specific to the PK gene family using cDNA derived from an OA patient as a template. Reaction conditions: 94 ° C (3 minutes)-[94 ° C (1 minute) -48 ° C (1 minute) -72 ° C (1 minute)] x using ExTaq polymerase (TaKaRa: RR001A) and its attached buffer 30 times-72 ° C. (5 minutes)), and the PK gene family fragment modified with biotin at both ends was specifically amplified. Hereinafter, the DNA obtained by this amplification is referred to as “driver DNA”.
(5) 2 μg of tester DNA for 6 patients and 4 μg of driver DNA for 3 patients were mixed and denatured by treatment at 100 ° C. for 5 minutes. An equal amount of hybridization buffer (6 × SSC, 5 × Denhardt solution, 0.1% SDS) was added to this solution, and incubated at 68 ° C. overnight to form a double strand between the tester DNA and the driver DNA.
(6) Add 500 μg of magnetic beads (Dynal Biotech; M-270 Streptavidin) to this reaction solution and stir for 1 hour to bind biotin in the driver DNA to streptavidin bound to the magnetic beads. I let you.
(7) The complex of driver DNA and magnetic beads was removed using a bead separation magnet (Dynal Biotech; MPC-S). As a result, not only the driver DNA but also the gene fragment that is common to the tester DNA and the driver DNA is removed. As a result, genes whose expression is increased in RA patients compared to OA patients remain in the solution.
(8) Driver DNA was newly added to the solution after the operation of (7), and the operations of (5) to (7) were repeated twice.
(9) In order to amplify the DNA contained in the solution after the operation of (8), PCR (reaction conditions: TAGAACTAGTGGATCC (SEQ ID NO: 7) and TCGACGGTATCGAT (SEQ ID NO: 8)) is used for the tester DNA amplification primers. 94 ℃ (3 minutes)-[94 ℃ (1 minute) -62 ℃ (1 minute) -72 ℃ (1 minute)] x 10-16 using ExTaq polymerase (TaKaRa: RR001A) and the attached buffer Times-72 ° C (5 min)).
(10) A fragment of about 240 bp, which is the fragment size of the PK gene family, was excised from the PCR product, and the fragment was subcloned into a plasmid vector using TOPO-TA Cloning Kit for Sequence (Invitrogen).
(11) The inserted sequence of the subcloned plasmid DNA was analyzed and searched in a database such as GeneBank. As a result, several types of PK genes were detected. Hereinafter, specific three types of detected PK genes are referred to as kinase A, kinase B, and kinase C, respectively.

<Example 2> Expression analysis in RA patients and OA patient synovium (1) To confirm that the expression of the PK gene identified by the subtraction method is increased in RA patients compared to OA patients In addition, PCR (reaction conditions: ExTaq polymerase (TaKaRa: RR001A)) and a buffer attached thereto using PCR from 6 RA patients and 3 OA patients as a template was used at 94 ° C. (3 minutes)-[94 ℃ (30 seconds) -60 ℃ (30 seconds) -72 ℃ (1 minute)] x 27 times -72 ℃ (5 minutes)). For amplification of each PK gene, PCR was performed using primers specific to each PK gene.
(2) PCR using a thermal cycler (TaKaRa; TP3000) using cDNA derived from RA and OA patients as a template, and 94 ° C (reaction condition: ExTaq polymerase (TaKaRa: RR001A) and its accompanying buffer. 3 min)-[94 ° C (30 sec) -60 ° C (30 sec) -72 ° C (1 min)] x 27 times -72 ° C (5 min)), then the PCR product was subjected to agarose gel electrophoresis The intensity of the corresponding band was quantified using an image analyzer (Bio-Rad; MOLECULAR IMAGER FX).
(3) The quantitative value of the band of each gene was calculated as a ratio to the quantitative value of G3PDH, which is an endogenous control, and the average value and standard deviation of RA and OA were determined. The quantitative value (expression level) of the kinase A gene is shown in FIG. 1, the quantitative value (expression level) of the kinase B gene is shown in FIG. 2, and the quantitative value (expression level) of the kinase C gene is shown in FIG.

As a result, the expression levels of kinase A, kinase B, and kinase C genes were higher in RA patients than in OA patients. From this, it was confirmed that the expression of the PK gene identified by the subtraction method was increased in RA patients compared to OA patients.

It is a figure which shows the expression level (RA, OA patient synovium) of a kinase A gene. It is a figure which shows the expression level (RA, OA patient synovium) of a kinase B gene. It is a figure which shows the expression level (RA, OA patient synovium) of a kinase C gene.

Claims (11)

A carrier for gene subtraction, wherein “nucleic acid holding nucleotide sequence to be annealed to“ nucleotide sequence common to gene family targeted for gene subtraction ”is bound. The carrier according to claim 1, wherein the gene family targeted for gene subtraction is a protein kinase gene family. The carrier according to claim 2, wherein the protein kinase gene family consists of a tyrosine kinase family and a serine / threonine kinase family. The carrier according to claim 2 or 3, wherein the "nucleotide sequence common to the gene family targeted for gene subtraction" is the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2.
GTGCACMGNGAYYT (SEQ ID NO: 1)
GAYGTSTGGTCCTWTGG (SEQ ID NO: 2)
(In the sequence number, M means A or C, Y means T or C, S means G or C, W means A or T, N means any one of A, G, C and T) (T may be U in the SEQ ID No. and in the description.) The nucleotide sequence annealed to "a nucleotide sequence common to a gene family targeted for gene subtraction" is the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 3, The carrier according to any one of claims.
GTGCACMGNGAYYT (SEQ ID NO: 1)
CCAWAGGACCASACRTC (SEQ ID NO: 3)
(In SEQ ID NO: M is A or C, Y is T or C, W is A or T, S is G or C, R is G or A, N is A, G, C and T (In addition, T may be U in the SEQ ID No. and in the present description.) The nucleotide sequence annealed to “a nucleotide sequence common to a gene family targeted for gene subtraction” is the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 4, The carrier according to any one of claims.
GTGCACMGNGAYYT (SEQ ID NO: 1)
GCGAATTCCAWAGGACCASACRTC (SEQ ID NO: 4)
(In SEQ ID NO: M is A or C, Y is T or C, W is A or T, S is G or C, R is G or A, N is A, G, C and T (In addition, T may be U in the SEQ ID No. and in the present description.) The carrier according to claim 1, wherein the carrier is a magnetic bead. The carrier according to any one of claims 1 to 7, wherein the binding mode between the nucleic acid and the carrier is a biotin-avidin bond. The carrier according to any one of claims 1 to 8, wherein the nucleic acid is DNA. A gene subtraction method using the carrier according to any one of claims 1 to 9. The “nucleic acid bound to the carrier according to any one of claims 1 to 9” and “the nucleic acid fraction containing the nucleic acid to be detected” are brought into contact with each other for hybridization, and then the carrier The gene subtraction method according to claim 10, further comprising a step of removing.

Images