JP2005117943A - Method for analyzing gene expression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing a gene expression, having high coverage and reproducibility. <P>SOLUTION: The method for producing a microbead, on which a DNA is immobilized, comprises (1) a process for fragmenting a sample-derived DNA, (2) a process for fractionating the fragmented DNA in size, (3) a process for linking the DNA fragment fractionated in size to a tag vector to prepare a tag library and (4) a process for preparing a DNA to which a tag sequence is connected from the tag library and connecting the DNA to a microbead. The method for analyzing a gene expression comprises using the microbead. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a gene expression analysis method using DNA microbead array technology.

  Details of the DNA microbead array technology have been reported by Brenner et al. (See, for example, Patent Document 1 and Non-Patent Document 1). In the above-described DNA microbead array technology, one target is obtained by hybridization between a single-stranded tag in which the portion from the most downstream GATC to the poly A sequence of the target DNA or cDNA is bound to an anti-tag bound on the bead. A single target DNA is associated with each microbead. The microbeads to which the target DNA is bound by hybridization are selected using a cell sorter. The target DNA is then covalently bound onto the beads by various enzymatic and chemical treatments. Furthermore, an adapter is added to the end of the target DNA away from the microbeads of the target DNA as necessary to make a single strand.

Japanese National Patent Publication No. 11-507528 Brenner S.M. , 12 others, Proceedings of National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 97, No. 4, pp. 1665-1670 (2000)

  In the microbeads described in Non-Patent Document 1, a cDNA fragment from the 3 ′ most GATC sequence (hereinafter sometimes simply referred to as GATC sequence) to a poly A sequence is immobilized as a target DNA. Depending on the presence or absence of the sequence, the distance from the poly A sequence to the GATC sequence, etc., there is a problem that the gene sequence fixed to the microbeads or the comprehensiveness of the gene to be analyzed for expression by Megasort is limited. Yes. For example, a gene that originally does not have a GATC sequence in cDNA is not cloned. In the case of a gene having a long distance from the poly A sequence to the GATC sequence, the synthesized cDNA may not contain the GATC sequence to the poly A sequence, and such cDNA is not cloned. In addition, a gene having a short distance from the poly A sequence to the GATC sequence has, for example, low specificity in competitive hybridization and may be excluded from the analysis target of megasort. Furthermore, since the immobilized cDNA is as short as 250 bp on average and is often an untranslated region, amino acid sequence information cannot often be obtained from the base sequence of DNA immobilized on beads.

  As a result of intensive studies, the present inventors have increased the fluorescence value at the time of megasorting by immobilizing size-fractionated DNA as a target DNA after fragmentation, and the comprehensiveness of genes targeted for megasort analysis. As a result, it was found that more sequence information can be obtained from the beads than the conventional microbeads, and the present invention has been completed.

Briefly describing the present invention, the first invention of the present invention is a method for producing microbeads on which DNA is immobilized, and includes the following steps.
(1) a step of fragmenting DNA derived from a sample;
(2) a step of size-fractionating fragmented DNA;
(3) linking the size-fractionated DNA fragment to a tag vector to produce a tag library; and (4) preparing a DNA to which a tag sequence is bound from the tag library and binding it to microbeads.

  In the first invention of the present invention, DNA can be fragmented by sonication in step (1). In step (2), a DNA fragment of 100 to 1000 bp is preferably recovered.

  As one aspect of the first invention of the present invention, a method of fragmenting and using cDNA prepared using mRNA as a template is exemplified. In this case, a 5'-side or 3'-side fragment of cDNA can be selected and ligated to a tag vector.

  A second invention of the present invention is a gene expression analysis method using microbeads on which DNA produced by the method of the first invention is immobilized, wherein the DNA on the microbead and two or more types And a step of performing hybridization with a nucleic acid probe labeled with a different label for each sample prepared from the RNA sample.

  In the method of the second invention of the present invention, a nucleic acid probe labeled with a fluorescent substance or a luminescent substance can be used.

  The present invention provides microbeads on which DNA having a certain range of chain length is immobilized.

The present invention will be specifically described below.
Terms used in the present specification will be described below. Unless otherwise defined, terms are used in the meaning generally understood by those skilled in the art such as molecular biology and molecular genetics.

  “DNA microbead array technology” means the technology disclosed in the above-mentioned Patent Document 1 and Non-Patent Document 1, and the technology for analyzing gene expression and gene structure by applying this technology. That is, this is a technique in which DNA to which an oligonucleotide called a tag is bound and microbeads to which an anti-tag, which is an oligonucleotide complementary to the tag, is bound by hybridization between the tag and the anti-tag. As a result, a library of DNA immobilized on microbeads can be prepared. Here, one type of DNA is bound to one microbead.

  A “tag library” means a library of DNA that is bound to microbeads in the DNA microbead array technology. Each clone of the tag library is DNA containing a tag sequence and DNA to be bound to microbeads, and a set of these clones is a tag library.

  A “tag” is an oligonucleotide that is covalently bound to DNA to be bound to microbeads in the DNA microbead array technology, and is used to place a single sequence of DNA on each microbead. The tag repertoire needs to be sufficiently larger than the number of tag library clones.

  The “anti-tag” is an oligonucleotide covalently bonded to microbeads in the DNA microbead array technology, and has a sequence complementary to the tag. A single sequence of anti-tags can be bound to a single microbead, and the anti-tag repertoire is substantially the same size as the tag repertoire.

  “Megasort” refers to the DNA on the microbeads that are competitively hybridized with two types of probes labeled with different dyes, such as Cy5 and fluorescein, and then the microbeads are combined with fluorescence intensity and dye. It is a technology that separates with a flow cytometer based on the ratio.

  “Target DNA” means DNA to be bound to microbeads and DNA bound to microbeads. The target DNA is not particularly limited, and may be, for example, the entire region of a gene encoding a certain polypeptide, or only a part thereof, for example, a 5'-side or 3'-side fragment.

  “E-value” is a numerical value representing the possibility that sequences that are not related to each other as a result of homology search will hit accidentally. The closer the e-value of the search result is to 0, the more the result can be regarded as a certain coincidence that is not accidental, and it can be said that it is statistically significant.

  “Specifically hybridize” means that a tag and an anti-tag that are complementary to each other hybridize, but a tag and anti-tag that contain a non-complementary sequence do not hybridize or hybridize only infrequently. means.

The gene expression analysis method of the present invention will be described in detail below.
(1) Step of fragmenting DNA derived from sample DNA prepared from a sample to be analyzed for gene expression state is fragmented. Examples of the DNA include, but are not limited to, genomic DNA, poly A RNA, and cDNA synthesized from total RNA. Those DNAs prepared by a known method can be used. As a more preferred embodiment, for example, in the case of cDNA, a portion corresponding to the 3 ′ or 5 ′ terminal side of the poly A RNA used as a template is subjected to a known method, for example, a biotin-labeled base or an adapter of a specific sequence There is a method in which each terminal-side cDNA fragment is selected after being prepared so that it can be discriminated using the binding of, and fragmented. This method is advantageous because it allows obtaining DNA fragments with clear direction information. The fragmentation method is not particularly limited, but may be a physical method (sonication, shear force load, etc.), a chemical method (acid treatment, etc.), an enzymatic method (by endonuclease such as DNase I). Decomposition etc.) can be used.

(2) Step of Size Fractionation of Fragmented DNA In this step, the DNA fragment obtained in (1) above is subjected to size fractionation, and a DNA fragment having an appropriate range of chain length is selected. The size fractionation method is not particularly limited, and any known method can be applied. For example, a method of recovering DNA from a gel after gel electrophoresis, a gel filtration method, an ultrafiltration method, or the like can be used. In this step, a fractionation operation is performed so that a DNA fragment having a chain length of 100 to 1000 bp, preferably 400 to 600 bp is recovered.

(3) Step of ligating the size-fractionated DNA fragment to a tag vector and preparing a tag library The fragmented DNA obtained in the above step (2) is ligated to the tag vector.
As the tag sequence contained in the tag vector, those disclosed in Patent Document 1 can be used, and among them, those described in Non-Patent Document 1 can be used particularly preferably. The tag vector preferably contains a selection marker such as drug resistance. When using Escherichia coli as a host, drug resistance genes such as ampicillin resistance, chloramphenicol resistance, kanamycin resistance, streptomycin resistance and the like can be used as markers.
The ends of the DNA fragments obtained in the above step (2) may be blunted before ligation to the tag vector. For the smoothing treatment, for example, treatment with BAL31 exonuclease, T4 DNA polymerase, Klenow enzyme, or a combination of these enzymes can be used. The DNA fragment thus prepared can be efficiently ligated to a linearized tag vector having a blunt end. The terminal shapes of DNA fragments fragmented by physical or chemical treatment are not uniform, and 5′-OH, 5′-phosphate, 3′-OH, and 3′-phosphate are mixed. Of these, only DNA having a 5′-phosphate group and a 3′-OH group is used as a substrate for a DNA-ligating enzyme such as T4 DNA ligase. Therefore, after removing the terminal phosphate group with an enzyme such as alkaline phosphatase If the 5 ′ end is selectively phosphorylated by an enzyme such as T4 polynucleotide kinase, a DNA fragment that can be efficiently ligated to a vector is obtained. The blunt end of the fragmented DNA can be converted to a protruding end by linking a linker or adapter. The DNA fragment to which the linker or adapter is linked may be directly linked to the tag vector, or may be linked after digestion with an appropriate restriction enzyme after amplification by PCR or the like, for example. This DNA fragment can be more efficiently ligated to a linear tag vector having a terminal complementary to the generated protruding terminal. At this time, when the DNA used in the above step (1) is processed so that each terminal can be identified, the DNA fragment on the desired terminal side is utilized, for example, using the affinity with streptavidin. After selection, a linker or adapter can be bound, or amplification can be performed by PCR using a primer having a specific sequence. The thus obtained DNA fragment can be inserted into the tag vector so that it is aligned in a certain direction reflecting the original DNA orientation with respect to the tag sequence.

A tag vector linked with fragmented DNA is introduced into a suitable host, preferably E. coli. Introduction of DNA into a host can be performed by a known method, and there is no particular limitation, but when transformation is performed using Escherichia coli as a host, an electroporation method or a method using competent cells can be used. Under the selection pressure corresponding to the marker gene of the tag vector, the transformant is cultured, and a tag library that is a mixture of the fragmented DNA and the DNA in which the tag vector is linked is prepared from the cells. In preparing the tag library, DNA may be prepared by a known method. When the tag vector is a plasmid vector, for example, alkali-SDS method can be used.
At this time, it is desirable to measure the number of independent clones contained in the tag library by inoculating a part of the transformed host cells into a solid selective medium and counting the number of colonies that appear.

The number of independent clones used in the following steps may be appropriately set depending on the purpose, but is set to 10 ⁴ or more, preferably 10 ⁵ or more, more preferably 10 ⁶ or more in consideration of the completeness of megasort. A method is effective in which a tag library is prepared as a plurality of pools and the desired number of clones is obtained by selecting the number of pools to be used. In this case, a tag library having the desired number of clones can be easily prepared by changing the number of clones per pool from 10,000 to several hundred thousand and the number of pools to be created from 2 to 100.

  The tag vector of Non-Patent Document 1 is a mixture of about 17 million types of plasmids having different tag portion sequences. In order to perform megasorting, it is necessary to bind one type of DNA to one microbead, and for that purpose, one type of tag DNA must correspond to one type of target DNA sequence. . By setting the number of clones contained in each pool of the tag library to 170,000 to 1,000,000 and binding the target DNA to the microbeads for each pool, the ratio of the tag to which two or more types of target DNAs are bound is determined. It can be suppressed to a low value of 0.5 to several percent.

When a tag library is prepared by the method of the present invention, for example, a gene that could not be cloned by the method of Non-Patent Document 1 because it has no GATC sequence, such as human ribosomal protein L37 mRNA, can be cloned. Thus, a cDNA fragment having a uniform chain length can be obtained without depending on the recognition sequence of the restriction enzyme. Further, according to the method of the present invention, for example, ribosomal protein L31 mRNA is obtained as a 428 bp cDNA fragment, and e-value is 0 when searched by homology search, for example, BLAST. In the method, since the gene can be obtained only as a short cDNA fragment of 23 bp, the e-value becomes 2e- ⁸ , and the reliability of the homology search is low.

(4) Step of preparing DNA to which tag sequence is bound from tag library and binding to microbeads From the tag library prepared in step (3), a fragment of DNA to which tag sequence is added is prepared. This method is not particularly limited. For example, after digesting the library with one or more appropriate restriction enzymes, the digested product is subjected to molecular weight fractionation by gel electrophoresis, gel filtration, or the like to obtain a vector portion. Examples include a method for preparing the removed DNA and a method using PCR. In particular, the PCR method has advantages in that it is simple when a large amount of target DNA and DNA containing a tag are prepared, and that only the target portion can be amplified, so that it is not necessary to remove the vector. In this case, the subsequent operation can be performed efficiently and easily by labeling the primer on the target DNA side with a fluorescent group and the primer on the tag side with a group capable of specific selection such as biotin.

  The tag portion of this DNA is made into a single strand by, for example, the following method. The tag of Non-Patent Document 1 consists of A, T, and G bases, and the complementary strand of the tag consists of T, A, and C. If necessary, the DNA is digested with a restriction enzyme that cuts between the tag and the tag-side end, the end fragment is removed, and then T4 DNA polymerase is allowed to act in the presence of dGTP so that only the tag portion is made into a single strand. it can.

  The target DNA fragment with a single-stranded tag thus obtained is bound to microbeads (hereinafter abbreviated as microbeads) bound to an anti-tag. Microbeads can be prepared, for example, by the methods described in Patent Document 1 and Non-Patent Document 1. Hybridization is carried out by mixing and incubating the target DNA with single-stranded tag and the microbeads. The conditions such as the composition of the incubation solution and the temperature are not particularly limited as long as the anti-tag on the microbead and the single-stranded tag bound to the fragmented target DNA specifically hybridize. As an example of conditions for specifically hybridizing, the present invention is not particularly limited, but the incubation solution described in Non-Patent Document 1, that is, 500 mM NaCl, 10 mM Na phosphate, 0.01% Tween 20, 3% dextran. A solution containing sulfuric acid is suitable for the present invention. The incubation time and temperature are not particularly limited, but the conditions described in Non-Patent Document 1 for hybridization at 72 ° C. for 3 days can be preferably used.

  After completion of the incubation, the microbeads are washed, and then the microbeads to which the single-stranded tagged target DNA has been bound by hybridization are selected. If the PCR primer on the target DNA side is fluorescently labeled, microbeads can be selected using a cell sorter using the label as an index, which is advantageous in terms of ease of operation, purity of the resulting microbeads, and the like.

  Next, a covalent bond is formed between the target DNA fragment bound to the microbead by the DNA ligase and the anti-tag. The DNA ligase to be used is not particularly limited, but for example, T4 DNA ligase and E. coli DNA ligase can be preferably used. Prior to the reaction with DNA ligase, if the microbeads are treated with DNA polymerase in the presence of dATP, dGTP, dCTP, or dTTP, the efficiency of the DNA ligase reaction may be improved. This reaction may be performed as necessary. Although there is no particular limitation on the DNA polymerase to be used, for example, T4 DNA polymerase can be preferably used.

  The DNA-immobilized microbeads produced by the above-described method have DNA having a specific range of chain lengths fixed irrespective of the position of the restriction enzyme site present in the gene. . It is very unlikely that only untranslated regions are immobilized on microbeads. Therefore, the microbeads thus obtained are very useful in comprehensive gene expression analysis.

Below, the gene expression analysis method using this microbead is demonstrated.
The gene expression analysis method of the present invention is based on the fluorescence or luminescent substance prepared from the DNA on the microbeads (target DNA-binding microbeads) prepared by the above method and two or more types of RNA samples. It includes a step of hybridizing with a labeled nucleic acid probe and reading fluorescence or luminescence of individual beads.

First, a DNA fragment containing a fluorescent group at the end of the target DNA present on the microbead is removed by restriction enzyme digestion. If necessary, an adapter may be added to recover the DNA immobilized thereon from the beads selected in the subsequent step.
Next, alkali treatment is performed to remove the DNA strand on the side where the anti-tag is not linked, so that the probe can hybridize to the target DNA on the beads.

  Any number of microbeads in which the target DNA portion is single-stranded may be used, but considering the completeness of the analysis target, it is desirable to probe more than 400,000 microbeads with a fluorescent dye. Hybridize DNA. Any number of probes may be used, but a fluorescent or luminescent labeled probe may be prepared from one type of sample and hybridized to a bead, or two or more types of samples may be labeled with separate fluorescent or luminescent dyes, Competitive hybridization can also be performed. The probe material is not particularly limited as long as it reflects the gene expression level. For example, cDNA, total RNA, polyA RNA, RNA prepared from cDNA by in vitro transcription, PCR product derived from cDNA library, etc. are used. do it. Although there is no limitation in particular in fluorescent dye, Cy5, Cy3, fluorescein etc. can be used, for example. Alternatively, for example, a primary-labeled DNA such as a biotin label can be used as a probe, and after hybridization, a fluorescent dye can be bound in two steps with, for example, phycoerythrin (PE) -labeled streptavidin. The probe labeling method is not particularly limited. For example, a PCR reverse transcription reaction, an enzymatic method of incorporating a fluorescent substrate during RNA synthesis / amplification using cDNA as a template, or a specific base such as a PCR product or cDNA may be used. A chemical method of fluorescent labeling may be used.

  The labeled probe and the target DNA-binding microbead are mixed and incubated, and hybridization is performed. The conditions such as the composition of the incubation solution and the temperature are not particularly limited as long as the probe specifically hybridizes to the single-stranded target DNA on the microbead. For example, the conditions described in Non-Patent Document 1, That is, conditions for hybridizing overnight at 65 ° C. in 4 × SSC, 0.1% SDS can be preferably used.

  After the incubation, the microbeads are washed, a region of interest is set as a gate by a cell sorter, and the microbeads contained therein are sorted. By cloning and sequence analysis of target DNA from the beads in the sorted region and performing clustering, homology search, etc., information on what number of genes are included in each gate can be acquired. The method for identifying the target DNA on the sorted microbeads is not particularly limited, but a method of amplifying the DNA fixed to the beads by PCR or the like and then cloning it into a plasmid vector to determine the base sequence can be suitably used. . The method for amplifying the target DNA on the microbeads is not particularly limited. For example, PCR using a plurality of beads as a template can be performed, or PCR can be performed after individual beads are separated. Alternatively, probe DNA or RNA hybridized with DNA on microbeads may be amplified by an appropriate method such as PCR or RT-PCR.

  For cloning of PCR products into vectors, use a method of ligating directly to a TA vector, etc., a method of ligating to a linearized vector with blunt ends after blunting the ends, and primers with appropriate restriction enzyme sites added The PCR product obtained in this manner can be digested with the restriction enzyme and inserted into a vector having a complementary end.

  According to the gene expression analysis method of the present invention, since the probe and the DNA on the microbead can be stably hybridized, it is possible to perform analysis with improved completeness and high reproducibility.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.
Among the operations described herein, the basic procedures such as plasmid DNA preparation and restriction enzyme digestion are described in Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition (Molecular Cloning-A Laboratory Manual-, Cold Spring Harbor Laboratory Press, 1989. Furthermore, E. coli TOP10 was used as a host in the following plasmid construction using E. coli unless otherwise specified. The transformed Escherichia coli is LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0) containing 30 μg / ml chloramphenicol, or 1.5% in the above medium. The cells were aerobically cultured at 37 ° C. using LB-chloramphenicol plates solidified by adding% agar.

Example 1
1. Sample Preparation HL-60 cells (ATCC CCL-240) were cultured at 37 ° C. in the presence of 5% CO _{2 in} RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1.3% dimethyl sulfoxide (DMSO). The cells were cultured for 7 days and differentiated into neutrophil-like cells. Further, after adding phosphate buffered saline (PBS) and culturing at 37 ° C. for 6 hours in the presence of 5% CO ₂ , a phorbol 12-myristate 13-acetate (TPA) solution having a final concentration of 100 ng / ml was added. The cells were cultured at 37 ° C. for 4 hours in the presence of 5% CO ₂ . Hereinafter, this cell is referred to as a TPA-treated cell. As a control, instead of giving TPA stimulation, a dimethyl sulfoxide (DMSO) solution was added and cultured in the same manner. Hereinafter referred to as control cells. TPA-treated cells and control cells were collected, total RNA was extracted with Trisol reagent (Gibco), and poly A RNA was further purified with Oligotex super (Takara Bio).

2. Tag library preparation HL-60 Reverse transcription reaction using 2.5 μg of poly A RNA derived from control cells as a template, consisting of a base sequence represented by SEQ ID NO: 1 in the sequence listing and a biotinylated 5 ′ end The double-stranded cDNA synthesized by the above was purified by phenol treatment, chloroform treatment and ethanol precipitation, and dissolved in 400 μl of TE buffer solution (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). The cDNA was sonicated four times for 15 seconds with UR-20P (Tomy Seiko), and then dephosphorylated with CIAP (calf intestine-derived alkaline phosphatase, Takara Bio). This DNA was purified by phenol treatment, chloroform treatment, and ethanol precipitation, and terminal blunting and phosphorylation were performed using a Takara Blunting ligation kit (manufactured by Takara Bio Inc.). DNA was separated by agarose gel electrophoresis, a gel block corresponding to a chain length of 400 to 600 bp was cut out, and DNA was extracted therefrom using QIAGEN gel purification kit (manufactured by Qiagen).

This DNA was bound to 1 mg of Dynabead M-280 streptavidin (magnetic streptavidin beads, manufactured by Dynal) for 30 minutes at 25 ° C., and left to stand for 30 seconds in MPC (manufactured by Dynal), and then the supernatant was removed. did. After washing the beads with 1 × BW buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2M NaCl), adapter DNA having a BamHI site was added, and T4 DNA ligase (manufactured by Takara Bio Inc.) The ligation reaction was carried out at 16 ° C. overnight. The adapter DNA consists of a base sequence represented by SEQ ID NO: 2 in the sequence listing and an annealed oligonucleotide with 5 ′ end biotinylated and the oligonucleotide represented by SEQ ID NO: 3. After washing the beads with 1 × BW, they were cut out from the beads with BsmBI (NEB), and the resulting DNA solution was purified by phenol treatment, chloroform treatment, and ethanol precipitation. Using this DNA as a template, PCR was performed using as a primer the biotinylated oligonucleotide represented by SEQ ID NO: 4 in the sequence listing and the oligonucleotide represented by SEQ ID NO: 5 in the sequence listing.
The PCR product was purified with Qiaquick PCR purification kit (Qiagen), digested with BamHI, bound to Dynabeads, and the recovered supernatant DNA was purified by phenol treatment, chloroform treatment and ethanol precipitation.

On the other hand, independently of the above method, cDNA fragments from the most downstream GATC sequence to the poly A sequence were obtained from TPA-treated cells and control cells according to the method of Non-Patent Document 1.
The tag vector pNCV2 described in Non-Patent Document 1 was digested with BamHI and BbsI (both manufactured by NEB), and then dephosphorylated with CIAP (Takara Bio).
Each of the cDNA fragment obtained by the method of the present invention using sonication and the cDNA fragment obtained by the method of Non-Patent Document 1 and the above-described linearized pNCV2 were treated with T4 DNA Ligase (Takara). E. coli TOP10 was transformed by electroporation using the obtained recombinant plasmid. A part of the transformant is inoculated on an LB-chloramphenicol plate, the number of clones is calculated from the number of colonies generated, and the remaining transformant is inoculated on an LB medium containing LB-chloramphenicol. Plasmid DNA was purified from a culture corresponding to 160,000 clones using QIAGEN Plasmid Midi Kit (Qiagen) to obtain a tag library consisting of 6 pools. When the nucleotide sequence of 96 clones of cDNA in the tag library was determined, the chain length of the inserted DNA was distributed in the range of 400 to 600 bp.

3. Preparation of microbeads 2. Using the plasmid pool prepared in step 1 as a template, PCR, digestion with restriction enzyme PacI (manufactured by NEB) and single-stranded tag portion with T4 DNA polymerase (manufactured by NEB) were performed by the method of Non-Patent Document 1.
50 μg of a single-stranded tagged target DNA fragment and 1.67 × 10 ⁷ anti-tag-bound microbeads were mixed and mixed in 100 μl of 500 mM NaCl, 10 mM sodium phosphate, 0.01% Tween 20, 3% dextran sulfate. Hybridization was performed at 72 ° C. for 3 days. The microbeads were washed with 50 mM Tris-HCl, pH 8, 50 mM NaCl, 3 mM MgCl ₂ followed by 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.01% Tween 20, and then a MoFlo cytometer (manufactured by Dakocytomation) was used. The microbeads with the top 1% fluorescence intensity by FAM were used for sorting (first sorting).

  A covalent bond was formed between the target DNA fragment and the anti-tag by allowing T4 DNA ligase to act on the sorted microbeads, and then digested with BamHI. Further, T4 DNA polymerase was allowed to act on the microbeads in the presence of dGTP, and then the adapter DNA was ligated using T4 DNA ligase. The adapter DNA is composed of an oligonucleotide represented by SEQ ID NO: 6 in the sequence listing and a base sequence represented by SEQ ID NO: 7 in the sequence listing and phosphorylated at the 5 ′ end and labeled with FAM at the 3 ′ end. Annealed with nucleotide. Finally, single strands were formed by alkali treatment, and microbeads without FAM fluorescence were sorted using a MoFlo cytometer (second sorting).

4). MegaSort (competitive hybridization)
M-MLV reverse transcriptase (TAKARA BIO INC.) Using 0.5 μg each of poly A RNA derived from control cells and TPA-treated cells as a template, and using an oligonucleotide with a T7 promoter sequence represented by SEQ ID NO: 8 in the sequence listing as a primer CDNA synthesis was carried out. Using this cDNA as a template, RNA was synthesized by T7 RNA polymerase (manufactured by Takara Bio Inc.). In addition, Fluorescein-dUTP was incorporated into cDNA derived from control cells, and Cy5-dUTP was incorporated into cDNA derived from TPA-stimulated cells to prepare RNA probes.

These probes were mixed with 400,000 microbeads (hereinafter referred to as conventional microbeads) prepared by the method of Non-Patent Document 1 from poly A RNA derived from control cells and TPA-treated cells, and a DIG Easy Hyb solution. Hybridization was carried out overnight at 50 ° C. (manufactured by Roche Diagnostics). On the other hand, said Example 1-3. The same hybridization was carried out on 400,000 microbeads prepared in the above (hereinafter referred to as microbeads of the present invention). After washing with 1 × SSC / 0.1% SDS and then with 1 × SSC / 0.1% SDS, analysis was performed using a MoFlo cytometer. As a result, the fluorescence value of the microbeads of the present invention was higher than that in the case of using the conventional microbeads, and was easily distinguished from the microbeads that did not hybridize.
Using a MoFlo cytometer, a region containing microbeads bound to genes whose expression levels were increased by TPA stimulation (hereinafter referred to as Up gate) and a region containing microbeads bound to genes whose expression levels were decreased (hereinafter referred to as “up gates”) , And Down gate), each 1% of the total number of microbeads was sorted.

5). Directional cloning
Microbeads were obtained by PCR using a part of the sorted beads, equivalent to about 200, as a template, and using the oligonucleotide shown in SEQ ID NO: 9 in the Sequence Listing and the oligonucleotide shown in SEQ ID NO: 10 in the Sequence Listing as primers. The above DNA was amplified. Takara ligation kit ver. 2 (manufactured by Takara Bio Inc.) was used to link to linearized pT7Blue2 (manufactured by Novagen) with T protruding one base on the 3 ′ side. For conventional microbeads, pT7Blue2 linearized with BamHI and NotI (both manufactured by Takara Bio Inc.) after digesting the PCR product amplified in the same manner with Sau3AI and NotI (both manufactured by Takara Bio Inc.). In the vector, Takara ligation kit ver. 2 was used for ligation. The clones derived from the conventional method microbeads and the microbeads of the present invention were subjected to sequence analysis of 192 each from the Up gate and the Down gate.

After clustering analysis using the Paracel clustering package (manufactured by Paracel), BLAST search was performed on ntDB. As a result, 102 hit genes were found. Among these, 6 types are genes that do not have a GATC sequence, such as ribosomal protein L37 mRNA (GenBank Accession No .: NM_0097), that is, genes that cannot be obtained by the method of Non-Patent Document 1. . In addition, 6 genes were obtained with a distance of 90 bp or less to the GATC sequence. As an example, ribosomal protein L31 mRNA (GenBank Accession No .: NM009932) having a distance of 23 bp from the GATC sequence to the poly A sequence can be mentioned. Even if the gene was obtained from conventional microbeads, the e-value at the time of BLAST search would be 2e- ⁸ , which would have been unreliable. However, according to the method of the present invention, the 3 ′ end region 428 bp of the gene is fixed to the microbeads, so that there is much sequence information obtained, and e-value is 0, with very high reliability, The gene could be identified.

When megasorting was performed using conventional microbeads, a gene having no GATC sequence could not be obtained. In addition, although three types of genes having a distance of 80 bp or less from the poly A sequence to the GATC sequence were obtained, all of them were short sequences, so the reliability by e-value in the BLAST search was low.
Therefore, immobilization of a fragment having a certain length on a bead regardless of the restriction enzyme site increases the specificity of hybridization in megasort, and further obtains longer sequence information. It is advantageous in that the gene can be identified with high reliability.
On the other hand, among the genes obtained with Megasort, the distance from poly A to GATC is long, for example, the gene of 500 bp or more is 10% of the total when conventional beads are used. According to the method, 22% of the total was obtained, and more genes having a long distance from the poly A sequence to the GATC sequence could be obtained.

Example 2
1. Sample Preparation Total RNA was extracted from the heart or skeletal muscle of 5 males and 5 females of mouse CL57BL with Trizol reagent (manufactured by Gibco).

2. Preparation of tag library cDNA was synthesized using 20 μg of the above total RNA as a template, and a chain length of 400 to 600 bp by sonication, dephosphorylation, smoothing and phosphorylation, and agarose gel electrophoresis in the same manner as in Example 1 above. DNA fragments corresponding to were collected and purified, and bound to avidin beads. Next, adapter DNA having an Sfa NI site inside was ligated. In addition, adapter DNA anneals the oligonucleotide shown by sequence number 11 of a sequence table, and the oligonucleotide shown by sequence number 12 of a sequence table. PCR was carried out using an avidin bead suspension containing the DNA linked with the adapter or a DNA solution cut out from the beads with BsmBI (NEB) as a template. The Sfa NI recognition sequence was inserted on the 5 ′ side of the primer on the poly A side of the cDNA.

A portion of the PCR product was digested with Sfa NI, which is a type IIs restriction enzyme, then bound to Dynabeads M-280 streptavidin (manufactured by Dynal), and the recovered supernatant DNA was treated with phenol, chloroform, and ethanol precipitated. Purified by
The fragmented DNA and pNCV2 linearized by the method of Non-Patent Document 1 were ligated with T4 DNA Ligase (Takara Bio), and E. coli TOP10 was transformed by electroporation using the resulting recombinant plasmid. did. Plasmid DNA was purified from a culture corresponding to 160,000 clones using QIAGEN Plasmid Midi Kit (Qiagen) to obtain a tag library consisting of 8 pools. When colony PCR was performed, the molecular weight of cDNA in the tag library was distributed in the range of 400 to 600 bp.

As a result of sequence analysis and clustering of arbitrary 96 clones from the library prepared from the mouse heart, 59 genes could be identified. Two of them are genes that do not have a GATC sequence, such as Mus musculus cytochrome oxidase, subunit VIc (Cox6c) mRNA (GenBank Accession No .: NM_053071), that is, genes that cannot be obtained by the method of Non-Patent Document 1. there were. In addition, three are genes having a distance of 90 bp or less from the poly A sequence to the GATC sequence. As an example, the gene is 58 bp, Mus musculus cytochrome subunit II (Cox2) mRNA complete cd (Gb | AF378830.1 | AF378830). Even if the gene was cloned by the method of Non-Patent Document 1, the e-value of the BLAST search result would have been 4e- ⁷ . However, according to the method of the present invention, since the gene has a fragment length of 467 bp, the e-value is 0, and gene identification can be performed with high reliability.

In addition, a gene having a distance of 500 bp or more from poly A to GATC may not reach GATC at the time of reverse transcription. For example, Mouse myosin heavy chain mRNA having a distance of 988 bp to GATC, 3 ′ blank . As shown in (GenBank Accession No .: M74751), four types of genes having a distance from the poly A sequence to the GATC sequence exceeding 500 bp were included.
From the above, unlike the conventional microbead array technology, it was possible to produce a tag library without being affected by the presence or absence of the GATC sequence or the position of the GATC sequence in the cDNA.

  The present invention provides microbeads on which DNA having a certain range of chain length is immobilized. The microbeads are excellent in gene coverage and hybridization stability with probes, and are useful in gene expression analysis in organisms. In particular, when the microbeads of the present invention are used in the megasort method, it is possible to increase the fluorescence value during megasort, increase the completeness of the megasort analysis target gene, and obtain more sequence information than the conventional method.

SEQ ID: 1; Primer to synthesizing cDNA from mRNA
SEQ ID: 2; Oligonucleotide containing adapter comprising BamHI site.
SEQ ID: 3; Oligonucleotide comprising adapter comprising BamHI site.
SEQ ID: 4; PCR primer.
SEQ ID: 5; PCR primer.
SEQ ID: 6; PCR primer.
SEQ ID: 7; Oligonucleotide cyclic adapter.
SEQ ID: 8; Primer to synthesizing cDNA from mRNA
SEQ ID: 9; PCR primer.
SEQ ID: 10; PCR primer.
SEQ ID: 11; Oligonucleotide comprising adapter comprising SfaNI site.
SEQ ID: 12; Oligonucleotide comprising adapter comprising SfaNI site.

Claims (7)

A method for producing microbeads having DNA immobilized thereon, comprising the following steps:
(1) a step of fragmenting DNA derived from a sample;
(2) a step of size-fractionating fragmented DNA;
(3) linking the size-fractionated DNA fragment to a tag vector to produce a tag library; and (4) preparing a DNA to which a tag sequence is bound from the tag library and binding it to microbeads. The production method according to claim 1, wherein in step (1), DNA fragmentation is performed by ultrasonic treatment. The method according to claim 1, wherein a DNA fragment of 100 to 1000 bp is recovered in step (2). 2. The production method according to claim 1, wherein the sample-derived DNA is cDNA prepared using mRNA as a template. The method according to claim 4, wherein in step (3), a 5 'or 3' fragment of cDNA is selected and ligated to a tag vector. A method for analyzing gene expression using microbeads, to which DNA is immobilized, produced by the method according to any one of claims 1 to 3, comprising DNA on the microbeads and two or more RNA samples. A method for analyzing gene expression, comprising a step of performing hybridization with a prepared nucleic acid probe labeled differently for each sample. The method for analyzing gene expression according to claim 6, wherein a nucleic acid probe labeled with a fluorescent substance or a luminescent substance is used.