KR100191894B1 - Detection method of sh2 segments gene by pcr - Google Patents

Abstract

The present invention relates to a method for searching for a new gene having a Sark-like region 2 (Src Homology 2, hereinafter referred to as SH2) from a cDNA library of human fetal liver tissue. The present invention relates to a new search method using polymerase chain reaction (PCR) which is simpler, can be carried out in a short amount of time, and does not use radioisotopes.

Description

Searching for genes with SH2 site using polymerase reaction

1 shows the results of a primary polymerase reaction search conducted according to the method of the present invention.

Molecular weight marker of pBR322 DNA digested with lane M. Hae III.

Lane 1. Amplification of Lck SH2 DNA by polymerase reaction.

Lanes 2-9. Polymerase product of cell eluate taken from each plate after primary plate culture.

2 shows the results of the secondary polymerase reaction conducted according to the method of the present invention.

3 shows the results of the tertiary synthetase reaction according to the method of the present invention.

The present invention relates to a method for searching for a new gene having a Sark-like region 2 (abbreviated as SH2) from a cDNA library of human fetal liver tissue. The present invention relates to a novel screening method using a polymerase chain reaction (PCR) which is simple, can be carried out in a short amount of time, and does not use radioisotopes.

More specifically, the present invention relates to SH2 from cDNA library of human fetal liver tissue by polymerase reaction using degenerate primers prepared from genetic information of several types of tyrosine kinase having known SH2 sites. The present invention relates to a method for finding a new gene having a site.

Cancer occurs when normal cells are genetically damaged by carcinogens such as viruses, chemicals, or radiation. More specifically, cancer is because the genetic information related to growth and proliferation is damaged, resulting in abnormal signaling pathways for intracellular growth.

Various proteins and polypeptides are involved in the process of delivering various signals transmitted from the outside of the cell into the cell nucleus. During this signaling process, receptors that receive external signals (epithelial growth factor, platelet-derived growth factor, etc.) are converted into double complexes by external signals, and tyrosine residues are phosphorylated and various signaling proteins that recognize phosphorylated tyrosine (force). Lipase Cr, SHC, phosphatidylinositol 3-kinase, Grb2, etc.) bind to these receptors. Such a signaling protein is characterized by having a site consisting of about 100 amino acids that can recognize a specific peptide of the phosphorylated receptor. In addition, the recognition site is called a sark-like site (SH2) because it is similar to c-Src, a tyrosine kinase in the cytoplasm.

The SH2 site was first discovered in 1986. (Sadowski, I., Stone, JC and Pawson, T., 1986., A noncatalytic domain conserved aming cytoplasmic protein-tyrosine kinase modifies the kinase function and transforming activity of Fujinami sarcoma virus p130gag-fps., Mol. Cell Biol. 6 : 4396-4408). SH2 sites recognize short amino acid sequences that contain phosphorylated tyrosine residues. Each signaling protein contains a different type and number of SH2 sites. It is a molecular switch that recognizes the peptide in the target protein and induces interaction between signaling proteins, and thus plays an important role in cell proliferation and differentiation. Therefore, among the various proteins involved in the signaling system related to the study of cancer and the treatment of cancer, we have developed inhibitors that can inhibit the binding of tyrosine phosphate, which is involved in the early stages, and signaling proteins including SH2 sites. There is much research going on. In particular, the analysis of the function of Src, a cancer gene of Ras' sarcoma virus (RSV), revealed that the protein function is protein tyrosine kinase. Since the function of the cancer gene product that causes endless proliferation of cells is tyrosine kinase, it has been confirmed that signaling on growth is activated or inactivated by tyrosine phosphorylation. In addition, after several normal growth factor receptors combine with external signals, the fact that the internal portion of the cell membrane of the receptor protein functions as a protein tyrosine kinase suggests that tyrosine phosphorylation is an essential way of growth signaling. have.

Specifically, specific binding at the SH2 site can be explained by the interaction of the site with amino acid residues immediately adjacent to the phosphorylated tyrosine residues. The specificity of the protein containing the SH2 site was further confirmed in 1993 by the structure of the Src SH2 site confirmed by X-ray structural crystallography. This structural determination revealed a mechanism of action for recognizing tyrosine residues in which the SH2 site was phosphorylated. As a result, it was found that specificity is indicated by the amino acid sequence of the protein including the SH2 site. In this study, we selected the most suitable phosphorylated peptide that binds to the Src SH2 site from a peptide mixture containing phosphorylated tyrosine residues and identified it as the pYEEI monomer. Specifically, this unit consists of glutamic acid of two polar and nonbasic residues and isoleucine of one large hydrophobic residue.

Peptides containing pYEEI monomers were used to identify the structure of the Src SH2 site and its bound form. The peptide binding site is a flat surface formed of two helices and one β-fold structure. In other words, the SH2 peptide complex is as if two protruding plugs (phosphorylated peptides) enter two recessed sockets (SH2 sites). The two holes in the SH2 site are very specific binding sites. One binds to tyrosine phosphorylated with ionic bonds, hydrogen bonds and hydrophobic bonds, and the other binds to isoleucine of pYEEI because it is primarily hydrophobic. The plug-in socket relationship between the SH2 site and the peptide is limited to a small range not exceeding 5-6 amino acid residues. This tight binding can explain the interaction between receptors containing various binding sites and different proteins, including SH2 sites.

As a result, it is possible to conquer cancer, which is the destiny of humankind, by clarifying the process of converting normal cells into cancer cells through a deep understanding of the initiation and continuous reaction of many signals transmitted from outside the cells described above. I think it's possible.

Molecular biological aspects of the cancer transition process are as follows: First, disruption of normal signaling due to abnormal cell growth stimulation delivery system and the activation of intracellular substances required. Second, disruption of intracellular growth balance by activation of cancer genes. It can be represented by an imbalance in cell growth regulation due to abnormal cycles.

Many initiation and subsequent reactions of intracellular signaling depend on the activity of protein kinases, which phosphorylate their substrates to elicit various physiological responses. In order for the various functions of cells to function normally, there must be the interaction of various organs, and this interaction starts with the stimulation and response by the proteins and growth factors produced in the cells. In particular, the ligand-receptor interaction is the starting point of intracellular signaling and ultimately delivers a signal to the cell nucleus to induce gene expression through the interaction of various signaling proteins. Through this process, the growth, metabolism and differentiation of cells are regulated by various proteins and other products produced by the expressed gene.

Such a series of intracellular actions is in most cases triggered by external stimuli, and these external signals are receptor tyrosine kinase, binding receptors with G protein, cytokine receptors, ion channels, serine threonine kinase receptors. And it is delivered intracellularly by chemokine receptors. Among them, most researches on receptor tyrosine kinase have been carried out, and about 20 types are known and 10 of them are related to cancer of cells, which is very important. The relationship between receptor tyrosine kinase (RTKs) and normal cell carcinogenesis has been studied in earnest with the discovery of receptor tyrosine kinase-activated cancer cell genes.

Thus, deep research into SH2, which plays an important role in the early stages of cellular signaling, facilitates the development of anticancer drugs. It is an object of the present invention to provide a simple, fast and accurate method for searching for a gene containing SH2, which is an important step in cancer research.

Genetic engineering retrieval of the library is a rapid analysis of whether a particular clone contains the desired nucleic acid sequence, which is first used to identify recombinant DNA clones in the library and to purify each clone [Frederick, MA, Roger , B., Robert, EK, David, DM, John, AS and Kevin, S. Screening Recombinant DNA Libraries. In: Short protocols in Molecular Biology (2nd eds.), 1992, pp 6 / 1-6 / 16].

Randomly cleaving the entire DNA of a chromosome containing a large number of genes, connecting the miscellaneous DNA fragments to a vector as a mixture, and then introducing them into the host microorganisms by transformation, yields a variety of clones with different DNA fragments. Since these clones contain the genes on the chromosome in their shape, they are called gene clones, and a population of such clones is called a genomic library. On the other hand, it is also possible to obtain a transfer RNA (mRNA) from a specific tissue or cell and enzymatically synthesize complementary DNA (cDNA) by a reverse electron enzyme in vitro, and clone it to obtain a complementary DNA clone. Can be. Such clone populations are called complementary DNA banks or complementary DNA libraries because they contain a variety of complementary DNA that reflects the purity of the transfer RNA used as a template.

Many studies have repeatedly used synthetic DNA technology to isolate specific genes or DNA fragments from an organism. But every time a new DNA fragment is needed, it is a time-consuming task to go through the entire cloning process. Thus, it is necessary to establish a collection of such bacteria that contains enough hybrid plasmids so that all DNA fragments in a cell appear once or in some cases twice. Searches using these aggregates are generally performed on bacterial clones or bacteriophage plaques, including plasmids or cosmids.

Among the methods of searching the library, the most commonly used method to obtain the nucleotide sequence of interest is the solubilization of the lysates formed by plate culture of the library using the phage as a vector to the nitrocellulose filter. After denaturation, hybridization is performed with DNA on the filter and radiolabeled DNA or oligonucleotide probes. Alternatively, complementary DNA libraries constructed from vectors that can be expressed by antibodies can be searched using antibodies, which are usually not sensitive enough to measure small amounts of delivery RNA (mRNA), or that false positives There are disadvantages that can come out. There is also a colony hybridization method, in which a large number of bacteria, each of which are thought to have interesting DNA, are immobilized directly on a nitrocellulose membrane to detect hybridized colonies with an autoradiograph using radiotransmitter RNA as a marker. to be. This method is a convenient method that can efficiently screen within a short time, but it is difficult to obtain high-purity transfer RNA that is used as a marker in practice. [Sambrook, J., Fritch, EF and Maniatis, T. Molecular Clloning: A Laboratory Manual (2nd ed). 1989. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.].

This hybridization method using a plate and a membrane several times to obtain a clone from the cDNA or gene library is time-consuming and experimental, as well as a number of fake wildness that is common in the filter hybridization method. Therefore, the present inventors studied a method of easily searching for a new gene having an SH2 site from cDNA or a gene library. By using the polymerase reaction method, positive clones were identified as DNA bands of the correct size on an agarose gel. The present invention has been completed and found to save a lot of time since it is possible to remove the false positive groups appearing in the conventional membrane hybridization method and also to use the appropriate primers to polymerize various kinds of genes at once.

Hereinafter, the present invention will be described in detail.

The polymerase reaction method of the present invention can be broadly divided into three steps. First, after culturing the library, adsorption and separation with a membrane, and then, the phage particles are washed from the second membrane, and the third phage eluate is performed for the synthesis of the enzyme. It is.

More specifically, phage libraries are plated with appropriate host bacteria to grow phage lysates and adsorbed onto nitrocellulose membranes, which are then transferred to a petri dish containing 3 ml phage dilution solution. Shake the membrane several times in the solution to wash the phage particles, leave for a few seconds, and then polymerase reaction with 20 μl of the phage eluate, the product was confirmed by agarose gel electrophoresis, at least one positive group in one culture plate When appears, the expected band will appear on the line on the gel. In this case, the screening can be continued through a general membrane hybridization method with a positive culture plate, and a search using a polymerase reaction can be continued. In addition, after adsorbing a nitrolocellulose membrane on a positive culture plate, the membrane is cut in 4-8 steps, placed on a gel, adsorbed, detached, and washed by the method described above. Also becomes smaller. The polymerase reaction is carried out with eluates from the washed fragments. If one of these fragments shows a positive result after the polymerase reaction, the above procedure is repeated.To re-plate the phage from the positive fragment, remove the top agar from a 50 ml conical tube containing 20 ml phage lime solution. After vigorous mixing, the supernatant is taken and the polymerase reaction or membrane hybridization is performed while controlling the amount and density of phage.

It will be described in more detail by the following examples.

The following examples illustrate the onset of disease and are not intended to limit the scope of the invention.

[I. Preparation and Search of cDNA Library in Human Fetal Liver Cells]

cDNA libraries were prepared from human fetal liver tissue using the λZAP cloning kit. DNA base sequence was determined using plasmid vector λZAP XR, and expression library was prepared using λZAP expression vector capable of expressing the cloned cDNA gene in eukaryotic cells.

Example 1 Isolation of RNA

RNA was isolated according to the AGPC method. Remove the fetal hepatocellular tissue stored in liquid nitrogen, place it in a sterile mortar, immediately pour liquid nitrogen, cut the tissue to the appropriate size, and then add solution D [4M guanidium cyanide, 25 mM sodium citrate, 0.5% sarcosyl ), 0.1M mercaptoethanol] and dissociated. Thereafter, the tissue was completely dissolved using a mesh (No. 60), and phenol and chloroform saturated with 2M aqueous sodium acetate solution were added at a 0.1: 1: 10.2 volume ratio, followed by centrifugation. The aqueous layer containing RNA was separated, transferred to a new test tube, and the RNA was precipitated with the same volume of isopropanol, stored at -20 ° C, dispensed as needed, centrifuged, and used.

Example 2 Purification of poly (A) + RNA]

An oligo (dT) cellulose column was prepared, and the sample RNA prepared in Example 1 was thermally denatured at 65 ° C. for 10 minutes and passed through the column with a buffer solution (20 mM Tris hydrochloric acid, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS). Poly (A) ⁺ RNA bound to cellulose was eluted with elution buffer (10 mM Tris hydrochloric acid, 1 mM EDTA, 0.05% SDS), and the poly (A) + RNA was recovered by precipitation with ethanol and centrifugation.

Example 3 Preparation of cDNA

A cDNA was prepared for cloning into a vector by mixing the poly (A) ⁺ RNA template and using the oligo (dT) 18-Xho I site GAGA sequence as a primer. Instead of dCTP in dNTP solution, methyl dCTP was added and reacted at 40 ° C. to obtain the first cDNA strand. After adding RNase H and DNA polymerase, the reaction was carried out at 16 ° C. for 1 hour and at 22 ° C. for 1 hour to synthesize a second cDNA strand. To determine the degree of cDNA synthesis, TD DNA polymerase and α ³² P-dCTP were added to the synthesized cDNA solution, followed by DNA synthesis.Some of the reaction solution was transferred to a 1.4% basic agarose gel (50mM NaOH, 1mM EDTA). It moved. After electrophoresis, the agarose was vacuum dried, and then exposed to a film for 3 hours to develop cDNA synthesis.

Example 4 Preparation of lambda phage library

After phosphorylating the cDNA prepared in Example 3, EcoRI / blunt adapter was added at a ratio of 1: 20-50, and then conjugated and phosphorylated overnight at 16 ° C. The base sequence of the used adapter is as follows.

Unconjugated adapters were removed via spin column with sephacryl S-400 and spliced at 16 ° C. overnight by mixing λZAP DNA treated with EcoRI and dephosphorylation CIP (calf intestinal phosphatase). Package Gold Plus was added to the reaction solution to carry out an in vivo package, and E. coli strain XL1-blue was selected to infiltrate each strain and observed the degree of formation of lysate plaques in the plate medium. At this time, cDNA fragments were inserted by adding X-gal and IPTG to determine the efficiency of the library by examining the ratios of white lysates that lost galactosidase activity and blue lysates without cDNA fragments. Escherichia coli cells for the formation of lytic plaques were cultured in LB medium containing 0.1% maltose and 10 mM MgSO ₄ to induce the expression of LamB functioning as λ phage adhesion proteins.The cells were recovered and suspended in 10mM MgSO ₄ solution and 600mm The absorbance at was used to be 0.5.

Example 5 Hybridization Process

Boehringermannheim's DIG labeling and detection kits were used for hybridization and detection.

(5-1) Marker of primer

The primers were labeled by polymerase reaction using UTP labeled with DIG. The primers used were as follows.

The reaction volume was adjusted to 50 μl by mixing 10 ng of DNA, 50 pmol of primer, 2.5 Unit of Taq polymerase, and then denatured for 5 minutes at 94 ° C, followed by 1 minute at 94 ° C, 50 seconds at 55 ° C, and 50 seconds at 72 ° C. The reaction was carried out 30 times in one time, followed by a polymerization reaction at 72 ° C. for 10 minutes.

(5-2) Preparation of the membrane

Cells stored in 96-well micro titer plates were transferred to 96 × 9 clones on a single membrane using a multi-transfer and incubated for 18 hours. After forming, a membrane for hybridization was prepared according to the following procedure. The 3MM membrane saturated with 0.5N NaOH, 1.5M NaCl, and 0.1% SDS solution was treated for 10 minutes with the membrane to be hybridized, and then the membrane denatured previously on the 3MM membrane saturated with buffer solution (1.0M Tris hydrochloric acid, 1.5M NaCL) was added. The DNA was fixed to the membrane by treatment twice a minute and then exposed to UV.

(5-3) hybridization

After 12cm × 7cm treatment with 200ml of prewash buffer (3 × SSC, 0.1% SDS) at 65 ° C. for 3 hours, 10ml of prehybridization buffer [5 × SSC, 1.0% blocking agent, 0.1 % N-laurylsarcosine] was added and subjected to prehybridization at 65 ° C. for 2 hours. After the membrane was treated, the same solution and a probe denatured for 10 minutes at 100 ° C. were sealed, and then reacted at 65 ° C. for 18 hours.

(5-4) washing and detection

After the hybridization reaction, the membrane was washed three times at room temperature with 2 × SSC 0.1% SDS solution for 20 minutes and at 65 ° C. for 20 minutes with 0.1 × SSC, 0.1% SDS solution. The detection method was followed by the following procedure.

The membrane was immersed in 10 ml of buffer 1 [100 mM maleic acid, 150 mM NaCl, pH 7.5], saturated for 5 minutes, and soaked in 10 ml of buffer 2 (buffer 1 + 1.0% blocker) for 60 minutes. After the reaction, buffer 2 was removed, and 1 μl of anti-DIG alkaline phosphatase was added to 10 ml of buffer 2, and the solution was left for 30 minutes. The reaction solution was removed and washed twice with 15 minutes of 1 + 0.3% Tween 20 solution. After washing, the mixture was saturated in buffer 3 [100 mM Tris hydrochloric acid (pH 9.5), 100 mM NaCl, 50 mM MgCl 2] for 5 minutes. Diluted with 10 ml buffer 3 (1: 100) Lumigen PPD (Lumigen PPD) solution was poured evenly to the membrane and allowed to react for 5 minutes at room temperature. After the reaction, the excess solution was removed, the film was wrapped in plastic wrap, the X-ray film was placed on the film, and the film was developed by sensitizing at room temperature for 20 minutes.

Example 6 Recovery of DNA from Clones of Library

λZAP, together with λDNA, contains pBluescript SK (-) pidmit DNA, which contains replication initiation and termination signal sites of line type viruses such as M13 or f1. With help, only pBluescript plasmids can be cut intracellularly. Replicated pBluescript is obtained as single-stranded DNA by ExAssist helper virus, infiltrated with E. coli strain SOLR ^TM , cultured in LB plate medium containing ampiciline, and then obtained a single colony containing kanamycin. The seed was added to the LB medium, and then a helper virus derived from M13K07 was added to obtain a single strand of phage, and then recovered by treating with 20% polyethylene glycol (PEG). The same volume of phenol was treated to remove proteins and precipitated with ethanol to purely separate single-stranded DNA.

[II. CDNA Library Search by PCR Method]

CDNA libraries prepared from human fetal liver tissue were searched through polymerase reaction using synthesized degenerate primers.

Example 7 Synthesis of Degenerate Primer

In order to conduct the polymerase reaction of this experiment, DNA base sequences of SH2 sites having the highest homology sites from five tyrosine kinase enzymes (lck, abl, hck, lyn, slk) were compared. A pair of primers were designed with a 22mer size as shown below and synthesized with a DNA synthesizer model 391 (Applied Biosystems, California, USA). The N-terminal region of the prepared primers had a 96-fold degeneracy and a conserved sequence of RK (D / A) AEWR (Q / L) L, and the C-terminal region had a 64-fold degeneracy. It has a conserved sequence of L (H / Q) (D / E) LV (R / Q) HY. The concentration of primers for the polymerase reaction was used so that each was 100 pmol.

Example 8 Primary Polymerase Reaction Search

1 × 10 ⁵ concentration of cDNA library was mixed with E. coli XL1-blue cell solution with absorbance of 1.0 at 660 nm and incubated at 37 ° C. for 30 minutes, then mixed with Top Agar and lysed at 50,000 lysis plates per plate on eight 150 mm culture plates. Incubation was carried out at 37 ° C. until they grew to adhere to each other. The cultured phages were soaked overnight at 4 ° C. with 10 ml of SM buffer solution, and then the SM buffer solution was collected from each plate and centrifuged to remove fragments at 7000 rpm for 10 minutes. Host E. coli was dissolved by adding 0.7% chloroform, and the phage stocks were stored at 4 ° C., and 1 μl of each phage stock was taken to perform a polymerase reaction at 50 μl.

The polymerase reaction was carried out as follows in a 50 μl reaction volume. 5 μl cell solution taken from each culture plate was used as a template, and the synthesized degenerate primer was 100 pmol, 200 μM of four deoxyribonucleotide triphosphates (dNTPs), and the mineral oil was dropped onto a mixed solution of 1.25 units of Taq polymerase. The polymerase reaction was carried out. The mixture was denatured at 95 ° C. for 5 minutes and then reacted 30 times with one time at 95 ° C. for 50 seconds, 60 ° C. for 50 seconds, and 72 ° C. for 1 minute, followed by polymerization at 72 ° C. for 5 minutes. The polymerase reaction was performed by DNA Thermal Cycler (Perkin Elmer Cetus, California, USA) and the results were analyzed on 2% MetaPhor agarose gel.

As shown in FIG. 1, a band was identified at 250 bp as a product of a synthesis enzyme amplified from lck SH2 DNA, which was performed as a comparative group in four culture plates among eight culture plates.

In the above method, since the extraction process of DNA for use as a template is not necessary, the search time is shortened, and any library can be searched by a polymerase reaction if only a small amount is present. Although the bacteria were fragments or inhibitors in the cell extract, they did not affect the method.

Example 9 Secondary Polymerase Reaction Search

Phage stocks selected as a result of the primary search in Example 8 were plated again to have a concentration of 500 plates of culture plate 500 on 8 plates of 100 mm size for the secondary search. Detailed culture method was the same as in the first polymerase reaction search of Example 8 and only the number of lysate plaques plated as described above was adjusted small. 4 ml of SM buffer solution was added to each culture plate and soaked overnight, followed by polymerase reaction in the same manner as in Example 8, and analyzed on a 2% MetaPhor agarose gel. As a result, as shown in FIG. 2, 250bp band was confirmed in 7 culture plates.

Example 10 Tertiary Polymerase Reaction Search

The positive collection solution obtained as a result of the secondary screening in Example 9 was re-platened to a concentration of 50 plates of culture plates. As a result of the procedure in Example 8 and Example 9, the same result as in FIG. 3 was obtained. Finally, the collection solution was plated again at a concentration of 50 solution plates, and several single solution plates generated in 200 μl SM buffer solution were eluted at 4 ° C. overnight. Phage insert DNA could be subcloned to determine the DNA base sequence of each eluate.

The method of searching a library using the polymerase reaction as described above has the following advantages as compared to the conventional search method.

First, the method using the polymerase reaction has an advantage of reducing the difficulty of searching before starting the existing membrane hybridization method. By pre-polymerizing the polymerase reaction with the entire library solution before searching for one library, it is easy to check whether the desired clone is in the library and to establish the presynthesis conditions that can amplify the desired signal.

You can also run multiple reputations simultaneously for library searches. For example, to retrieve 106 fibrous platelets, 20 plates are needed to form 50,000 platelets per plate. The screening of these plates with conventional membrane hybridization methods takes considerable time and effort, but the synthesis enzyme detection method takes only a few hours. Furthermore, the positive plate can be cut into eight sections for secondary retrieval, and they can also be used as a common membrane hybridization method. In addition, phage eluates can be supplemented to search for different genes with the same reputation. In order to search for genes by polymerase reaction, amplification of genes with small gene size is more than 30 times. When amplifying genes using polymerase reaction in this way, they are searched several times without losing the positive signal of the plate laid down due to the purity. The plate can be stored for several weeks at 4 ° C before lifting the lift.

Since the method of the present invention directly uses the phage eluate, no DNA extraction process is required, so that a large number of samples can be easily searched, and thus, the cost of the search system is low. It does not require a nitrocellulose membrane and is less toxic because no radioisotopes and X-ray films are used. It also saves time by eliminating the need for a hybridization process, cleaning process, and waiting time for photosensing, thus making this method simpler and better than all the conventional methods previously described.

Claims (5)

1) adsorption and separation of the library after plate culture, 2) washing the phage particles from the membrane to prepare phage eluate 3) phage eluate and five types of tyrosine kinase (lck, abl, hck, lyn) , slk) performing a polymerase reaction using one or more of the degenerate primers having the following nucleotide sequence of 22mer size prepared by comparing the SH2 DNA base sequence of the region having the highest homology region; 4) A method for searching for a gene having a SH2 site using a polymerase reaction, characterized in that the product consists of agarose gel electrophoresis. The method of claim 1, wherein the library is a cDNA library of human fetal liver cell tissue. The method according to claim 1, wherein the polymerase reaction is carried out directly with the cell eluate without separating and purifying DNA after plate culture. The search method according to claim 1, wherein after the step 4), the search can be further performed through a general film hybridization method. The search method according to claim 1, wherein after the step 4), the search can be performed using the polymerase reaction continuously.