JP2003279568A - Method for detecting bonding region of dna binding protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a binding region of DNA binding proteins, which eliminates the need to newly produce a DNA micro-array for the region between genes and suppresses experimental errors. <P>SOLUTION: A genome DNA 10 to which marked DNA binding proteins X, Y and Z are bonded, is cut into a plurality of DNA fragments, and then these fragments are hybridized with the DNA micro-arrays 20-40 where the genes b1-b6 are spotted. The hybridization is detected at two spots on the DNA micro-array, and thereby making a judgement that the binding region of the DNA binding protein exists in the two regions between genes. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a DNA region of a gene to which a DNA-binding protein specifically binds, using a DNA microarray.

[0002]

Transcription of genes on genomic DNA is controlled by transcription factors. The transcription factor is D upstream of the gene.
There are a DNA-binding protein that specifically binds to the NA region and a protein that does not directly bind to DNA but acts through factor interaction. A method using a DNA microarray is known as a method for detecting a DNA region upstream of a gene to which a DNA-binding protein specifically binds ("Genomic bin
ding sites of the yeast cell-cycle transcription f
actors SBF and MBF ", Nature, vol.409 (25 January 2
001), pp.533-538).

FIG. 6 is an explanatory view of this conventional method. First, a probe is prepared. In the conventional method, as shown in FIG.
As shown in (a), the DNA sequences a1, a2, a3, a4, a5 of the intergenic regions are obtained from the genomic DNA 10 composed of the genes b1, b2, ... And the intergenic regions a1, a2 ,. It is spotted on a slide glass to prepare a plurality of DNA microarrays 50, 60, 70 as shown in FIG. 6 (c). The displays a1, a2, ... In FIG. 6 (c) are DNA microarrays 50, 6
DNA sequences a1, a2, ...
Represents a fixed spot.

Next, a target is prepared. Figure 6 (b)
As shown in (3), the tagged transcription factors X, Y, and Z are cross-linked after binding to the gene expression induction site of the genomic DNA 10 for detection. Then, this genomic DNA 10 is cut into pieces with an ultrasonic disrupter and the like, and only DNA fragments to which the tagged transcription factors X, Y, Z are bound are extracted by immunoprecipitation. Furthermore, after removing the cross-links and separating the transcription factor and the DNA fragment, the DNA fragment is fluorescently labeled as targets X, Y, and Z.

Next, as shown in FIG. 6C, the DNA sequences a1 and a on the DNA microarrays 50, 60 and 70 are formed.
The targets X, Y, and Z prepared previously are hybridized with the spots immobilized with 2, a3, a4, and a5 as probes. At this time, the DNA microarray 50
The target X to which the transcription factor X was bound was reacted on top. Similarly, the target Y was made to react on the DNA microarray 60, and the target Z was made to react on the DNA microarray 70.

On the DNA microarray 50 of FIG. 6 (c), the DNA sequence a1 hybridizes with the target X, and as a result, the transcription factor X is an intergenic region on the genomic DNA 10 which is a region between the genes b1 and b2. a1
It turns out that it binds to. Similarly, since the DNA sequence a3 and the target Y react with each other on the DNA microarray 60, the transcription factor Y is a region between the gene b3 and the gene b4 on the genomic DNA 10, which is an intergenic region a.
It turns out that it binds to 3. DNA microarray 70
It can be seen that the transcription factor Z binds to the intergenic region a5 between the gene b5 and the gene b6 because the DNA sequence a5 and the target Z react with each other.

[0007]

In a conventional method for detecting a binding site of a DNA-binding protein using a DNA microarray, as shown in FIG. 6 (a), all the sequences of the intergenic region are prepared and the spotted DNA is prepared. It is necessary to make a new microarray. Further, in the detection method using the DNA microarray, an experimental error is likely to occur depending on the conditions at the time of hybridization. For example, according to the above-mentioned conventional method, the array image data of the DNA microarray 50 reacted with the target X is read by a scanner, the abscissa indicates the intergenic region name, and the ordinate indicates the fluorescence intensity of the spot. In some cases, the result shown in FIG. 7 may be obtained. In FIG. 7, DNA sequences a1 and D
The data of the NA array a5 exceeds the specified value. In this case, the fluorescence intensity of the DNA sequence a5 exceeds the threshold value due to an experimental error, but it is not possible to judge which is the correct data and which is the experimental error only from the result of FIG. 7. The present invention has been made in view of the above problems of the prior art, and it is an object of the present invention to provide a method for detecting a binding region of a DNA-binding protein that does not require the production of a new intergenic region DNA microarray and is less likely to cause an experimental error.

[0008]

[Means for Solving the Problems] A plurality of genomic DNAs obtained by binding and cross-linking a tagged DNA-binding protein are prepared.
After cutting into NA fragments, only the desired DNA fragment is extracted by immunoprecipitation. The cross-link is removed, and the DNA fragment is fluorescently labeled as a target. At this time, the target includes the intergenic region and a part of the genes at both ends. DN where this target is spotted with "DNA sequence of gene" instead of "DNA sequence of intergenic region"
Hybridize to A microarray. The target is detected at two spots on the DNA microarray. From this, it can be detected that the binding region of the DNA binding protein exists in the intergenic region of the two genes.

A method for detecting a binding region of a DNA binding protein according to the present invention is a method in which a plurality of probes are spotted on a substrate.
In a method for detecting a binding region of a DNA-binding protein, which detects an intergenic region of genomic DNA to which a DNA-binding protein binds using NA microarray, genomic DNA is used as a plurality of probes spotted on the DNA microarray.
Using a plurality of genes in it, as a target, at least a part of the intergenic region to which the DNA-binding protein binds,
DNA containing at least a part of a gene adjacent to an intergenic region to which a DNA binding protein binds on genomic DNA
It is characterized by using fragments.

The method for detecting a binding region of a DNA binding protein according to the present invention also provides a method for detecting a binding region of a DNA binding protein for detecting an intergenic region of genomic DNA to which the DNA binding protein binds. Preparing a DNA fragment containing at least a part of the intergenic region of DNA and at least a part of the gene adjacent to the intergenic region on the genomic DNA;
A step of performing a hybridization reaction between the immobilized gene and the DNA fragment using a DNA microarray in which a plurality of genes contained in genomic DNA are immobilized at different positions; and a plurality of genes immobilized in the DNA microarray Detecting the gene to which the DNA fragment is bound, and the intergenic region flanking the two genes bound to the DNA fragment on the genomic DNA and sandwiched by the two genes is DNA.
Determining the intergenic region to which the binding protein binds.

In the case of a bacterial genome, the target DNA fragment has a length of the intergenic region of 300-8.
Since it is 00, it is preferably 500 to 1500 bases in order to include a part of the genes at both ends. On the other hand, in the case of the genome of higher organisms, since the length of the intergenic region is several k to several tens of k, it is necessary to set the target length to 1 k to 100 k. In other words, since the distance between genes differs depending on the organism used as a probe, it is necessary to change the length of the target to be created (The Sequence of the Human Genome
: Science Vol.291, No.5507, Issue of 16 Feb 2001,
pp. 1304-1351.).

DNA reacted with DNA microarray
The fragment includes a DNA fragment containing at least a part of a first gene adjacent to the upstream side of an intergenic region of genomic DNA to which a DNA binding protein binds, and at least a part of a second gene adjacent to the downstream side. DNA fragment.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Here, a DNA fragment attached to a DNA microarray is used as a probe, and a hybridization reaction is performed with the probe.
The NA fragment will be called the target.

FIG. 1 is a flow chart showing the procedure of a method for detecting a binding site of a DNA binding protein (transcription factors X, Y, Z) according to the present invention. FIG. 2 is an explanatory diagram of each step. In step 10, a DNA microarray is prepared. As shown in FIG. 2A, the genomic DNA 10 is composed of genes b1, b2, b3, b4, b5, b6 and intergenic regions a1, a2, a3, a4, a5. Here, DNs of genes b1, b2, b3, b4, b5, b6
Obtain the A sequence as a probe. In the case where all genes have been determined as in yeast, cDNA of the gene is prepared using primers, and this is amplified by PCR to be used as a probe. By spotting the probes on the slide glass, multiple DNA microarrays 2
0, 30, 40 are produced.

In the next step 11, a target is prepared. First, the transcription factors X, Y, and Z with a tag as shown in FIG. 2B are cross-linked after binding to the genomic DNA 10. Then, the genomic DNA to which the tagged transcription factors X, Y, Z are bound is crushed by an ultrasonic crusher, and the transcription factors X, Y,
Based on the Z tag, DNA fragments are separated and extracted by immunoprecipitation. Furthermore, after removing the crosslinks and separating the transcription factor and the DNA fragment, the DNA fragment is fluorescently labeled as targets X, Y, and Z. At this time, in the conventional method of extracting only the intergenic region, it was cut to 300 to 800 bp, but in the present invention, about 500 to 1500 bp,
Cleavage is preferably performed at about 1000 bp (in the case of bacterial genome). This allows the DNA fragment to include not only the intergenic region but also a part of the genes on both sides.

In the case of the genome of higher organisms, since the length of the intergenic region is several k to several tens k, the target should be 1 k to 100 k in order to include a part of the gene at both ends. There is a need. In other words, the distance between genes differs depending on the organism used as a probe, so it is necessary to change the length of the target to be created.

FIG. 3 is an explanatory view showing a set of DNA fragments separated by the same tag. Here, the tagged transcription factor X bound to the intergenic region of gene b1 and gene b2 is shown. The separated DNA fragments include those shown in the figure, which are targeted. D
NA fragment I contains a part b1 ′ of gene b1 and a part b2 ′ of gene b2 at both ends, and intergenic region X is present in the center. The DNA fragment II contains a part X ′ of the intergenic region X and a part b2 ′ of the gene b2. The DNA fragment III contains a part b1 ′ of the gene b1 and a part X ′ of the intergenic region X. The DNA fragment IV contains a part X ′ of the intergenic region X.

Next, in step 12, step 1
The target prepared in step 11 is sprinkled on the DNA microarray prepared in 0 to carry out a hybridization reaction. At this time, the DNA fragment I shown in FIG.
The target composed of II and III hybridizes with the probe on the slide glass as shown in FIG. That is, the target composed of the DNA fragment I shown in FIG. 3 hybridizes with the probe composed of the gene b1 or the probe composed of the gene b2. DNA fragment II shown in FIG.
Target consisting of hybridizes with a probe consisting of gene b2. Similarly, the DNA fragment shown in FIG.
The target composed of III hybridizes with the probe composed of the gene b1.

Further, in FIG. 2 (c), the target X (DNA fragment b1 'is set to the DNA microarray 20).
-X-b2 ') was reacted. Here, the b1 ′ part of the DNA fragment b1′-X-b2 ′ is hybridized to the probe composed of the gene b1, and the b fragment of the DNA fragment b1′-X-b2 ′ is hybridized to the probe composed of the gene b2.
The 2'portion is hybridized. Similarly, target Y (DNA fragment b3 '
-Y-b4 ') was reacted. At this time, the b3 'portion of the DNA fragment b3'-Y-b4' hybridizes to the probe sounding from the gene b3, and the b4 'portion of the DNA fragment b3'-Y-b4' hybridizes to the probe consisting of the gene b4. It is soybean. Furthermore, target Z (DNA fragment b5′-Z-b
6 ') was reacted. At this time, the b5 'portion of the DNA fragment b5'-Z-b6' was hybridized to the probe composed of the gene b5, and the b6 'portion of the DNA fragment b5'-Z-b6' was hybridized to the probe composed of the gene b6. Are hybridizing.

In the next step 13, the transcription factor binding position is determined by examining the result obtained in step 12. Now, the result of the hybridization reaction of the DNA microarray 20 shown in FIG. 2 (c) is read as image data by a scanner, and a bar graph with the gene name on the horizontal axis and the fluorescence intensity on the vertical axis is prepared as shown in FIG. However, it is assumed that the data of b1, b2, and b5 exceed the threshold value. The intergenic region of interest to which the DNA binding protein binds exists between adjacent genes. And
According to the method of the present invention, the target should hybridize with a pair of probes consisting of adjacent genes in genomic DNA. Gene b exceeding the threshold in FIG.
Among 1, 1, b2 and b5, b1 and b2 are adjacent to each other on the genomic DNA. Therefore, from the result of FIG. 5, b5
Is based on experimental error, and transcription factor X is gene b
It can be seen that it is bound between 1 and gene b2.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, it is not necessary to prepare all intergenic regions of genomic DNA and spot them on a DNA microarray, and a commercially available D on which genes are spotted is used.
NA microarrays are available. This eliminates the need to newly create a DNA microarray in the intergenic region, and is expected to reduce costs. Further, by observing the reaction of two probes composed of adjacent genes on the genomic DNA, the error that occurs during the hybridization reaction can be eliminated, so that an experimental error is less likely to occur.

[Brief description of drawings]

FIG. 1 is a flow chart showing the procedure of a method for detecting a binding site of a DNA binding protein according to the present invention.

FIG. 2 is an explanatory diagram of each step in FIG.

FIG. 3 is an explanatory view showing a set of DNA fragments separated by the same tag.

FIG. 4 is an image diagram of a hybridization reaction.

FIG. 5 is a view showing a reaction result of a DNA microarray.

FIG. 6 is an explanatory diagram of a conventional method.

FIG. 7 is a diagram showing the reaction results of a DNA microarray.

[Explanation of symbols]

10 ... Genomic DNA, 20-40 ... DNA microarray, 50-70 ... DNA microarray, a1-a5 ...
Intergenic region, b1 to b5 ... Gene, X, Y, Z ... Transcription factor and target

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tsunehiko Watanabe             6-81 Onoe-cho, Naka-ku, Yokohama-shi, Kanagawa             Hitachi Software Engineering Stock Association             In-house (72) Iwa Yamashita, Inventor             6-81 Onoe-cho, Naka-ku, Yokohama-shi, Kanagawa             Hitachi Software Engineering Stock Association             In-house (72) Inventor Takuro Tamura             6-81 Onoe-cho, Naka-ku, Yokohama-shi, Kanagawa             Hitachi Software Engineering Stock Association             In-house F-term (reference) 4B024 AA11 BA80 CA01 CA09 CA11                       HA12                 4B063 QA01 QA18 QQ42 QQ52 QR08                       QR42 QR55 QR62 QR82 QS25                       QS34 QS36 QX02

Claims (5)

[Claims] 1. A method for detecting a binding region of a DNA-binding protein, which comprises detecting an intergenic region of genomic DNA to which a DNA-binding protein binds by using a DNA microarray in which a plurality of probes are spotted on a substrate. Using a plurality of genes in the genomic DNA as a plurality of probes, and as a target, at least a part of the intergenic region to which the DNA binding protein binds, and the intergenic region to which the DNA binding protein binds on the genomic DNA A method for detecting a binding region of a DNA-binding protein, which comprises using a DNA fragment containing at least a part of a gene adjacent to DNA. 2. Genomic DN to which a DNA-binding protein binds
A method for detecting a binding region of a DNA-binding protein for detecting an intergenic region of A, wherein at least a part of the intergenic region of the genomic DNA to which the DNA binding protein binds and the adjacent intergenic region on the genomic DNA A step of preparing a DNA fragment containing at least a part of the gene; and a step of preparing the immobilized gene and the DNA fragment by using a DNA microarray in which a plurality of genes contained in the genomic DNA are immobilized at different positions. Performing a hybridization reaction, detecting a gene to which the DNA fragment is bound among the plurality of genes immobilized on the DNA microarray, and adjoining the two genes to which the DNA fragment is bound on the genomic DNA. The intergenic region flanked by the two genes A method for detecting a binding region of a DNA-binding protein, comprising the step of determining an intergenic region to which the DNA-binding protein binds. 3. The method for detecting a binding region of a DNA binding protein according to claim 2, wherein the length of the DNA fragment is 500.
˜1500 bases, a method for detecting a binding region of a DNA binding protein. 4. The method for detecting a binding region of a DNA binding protein according to claim 2, wherein the DNA fragment has a length of 1 k to
A method for detecting a binding region of a DNA binding protein, which has 100 k bases. 5. The method for detecting a binding region of a DNA binding protein according to claim 2, wherein the DNA fragment is the DNA
A DNA fragment containing at least a part of a first gene adjacent to the upstream side of the intergenic region of the genomic DNA to which a binding protein binds, and a DNA fragment containing at least a part of the second gene adjacent to the downstream side A method for detecting a binding region of a DNA-binding protein, which comprises: