WO1998023736A1 - Procede d'isolement d'un gene apparente a un phenotype, region de transition ainsi que transition moleculaire de ce gene - Google Patents

Procede d'isolement d'un gene apparente a un phenotype, region de transition ainsi que transition moleculaire de ce gene Download PDF

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WO1998023736A1
WO1998023736A1 PCT/CN1997/000136 CN9700136W WO9823736A1 WO 1998023736 A1 WO1998023736 A1 WO 1998023736A1 CN 9700136 W CN9700136 W CN 9700136W WO 9823736 A1 WO9823736 A1 WO 9823736A1
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fragments
gene
dna
genes
fragment
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PCT/CN1997/000136
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Chinese (zh)
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Xiaozhuang Bian
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Xiaozhuang Bian
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Priority claimed from CN96119585A external-priority patent/CN1057338C/zh
Priority claimed from CN 97109139 external-priority patent/CN1167149A/zh
Application filed by Xiaozhuang Bian filed Critical Xiaozhuang Bian
Priority to AU51151/98A priority Critical patent/AU5115198A/en
Publication of WO1998023736A1 publication Critical patent/WO1998023736A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • the present invention relates to a method and an apparatus for isolating a DNA fragment of a phenotype-related gene from a complex genome of a human or a higher plant or animal having a certain phenotype.
  • it relates to a method and a device for directly and accurately separating DNA fragments of any type and any type of phenotypic genes in the whole genome DNA strand.
  • the present invention also relates to the use of known persuasive gene probes to screen natural switch molecules that modify the gene to change its expression state, thereby establishing a method for controlling target gene expression according to natural procedures or simulating natural procedures.
  • it relates to a method capable of efficiently and accurately inactivating harmful genes such as human pathogenic genes, activating human disease control genes and beneficial genes such as economic animals, high-yield plants, high quality, disease resistance, and stress resistance. Background technique
  • Phenotypic-related gene fragments include multiple types, such as blood-same isotype fragments or isogenic lines in a lineage. (Somatic cell lines of the same individual of higher organisms, monoclonal lines of plants and monoclonal lines of animal and plant cells); mutations in rearrangements (gene deletions, insertions, inversions, etc.), point mutations (base substitution and Small deletions, insertions) or demethylated mutant fragments; single genotype mutations or multiple genotype mutation fragments.
  • mutations in rearrangements gene deletions, insertions, inversions, etc.
  • point mutations base substitution and Small deletions, insertions
  • demethylated mutant fragments single genotype mutations or multiple genotype mutation fragments.
  • the methods of isolating genes can be divided into two categories: (1) Selective isolation of genomes by specific probe labeling (hybridization) or specific primer PCR amplification Genes in some regions. Such methods reduce the complexity of the genome at the cost of reducing the area of the genome being screened, which will inevitably reduce its sensitivity (N. Lisitsyn, et al., Science, 259 (1993), P946-951; P. Liang, et al "Nucl. Acid. Res. 21 (1993), P3269-3275).
  • Antisense mRNAs have a short half-grief period and are difficult to enter the cells; moreover, these substances only act for a short time and cannot permanently (from DNA) change the gene expression state (phenotype).
  • the existing technologies for changing gene expression states are all unnatural methods, which are low in efficiency, limited in accuracy, and difficult to put into practice. Therefore, until now, the technology of regulating the expression of eukaryotic genes has yet to be broken. A method for efficiently and precisely regulating gene expression using natural procedures is needed. Summary of invention
  • One of the present invention is to improve the above-mentioned defects existing in the existing phenotypic cloning method, and to establish a method that can directly and accurately isolate any type of phenotypic related genes in any part of the entire genome.
  • Two-dimensional electrophoresis was used to replace the existing one-way electrophoresis method before hybridization, and two-dimensional electrophoresis (coarse cut and fine cut) gel filtration electrophoresis was used to replace the existing two-dimensional electrophoresis before band comparison.
  • the complexity of the genome has been reduced to less than 10 2 to the point where it can be directly distinguished.
  • Subtraction hybridization or banding comparison of mutants in the whole genome that can separate rearranged mutant fragments is achieved. Isolation of any type of mutant fragments in the genome is achieved.
  • the present invention relates to a method for isolating phenotype-related genes, which includes the following steps: In the first step, a sample and a control DNA fragment are dispersed on a gel, thereby reducing the number of DNA fragments overlapping at the same site. Below 10 2 ; in the second step, the gel sheet is processed to hybridize the DNA fragments contained therein in situ by electrophoresis; in the third step, non-hybridized rearranged mutant fragments and demethylated / methylated The mutant fragment and the point mutation fragment that hybridizes to form a mismatch are separated on another membrane. In the fourth step, the mutant fragment, ie, the phenotype-related gene fragment of the isogenic line, is in situ.
  • PCR or PCR amplification in a test tube as a probe after autoradiography
  • PCR amplification of a non-mutated fragment that is, a phenotype-related gene fragment of a genetic line, in a test tube as a probe after autoradiography
  • the sample and the control fragment are mixed and electrophoresed so that the control fragments are fully hybridized, and the two-dimensional gel filtration electrophoresis is used to displace the non-control fragments without complexity.
  • the present invention also relates to a device for isolating phenotypic-related genes, which can be used to implement the above method, which comprises a three-dimensional electrophoresis apparatus for two-dimensional electrophoresis of DNA fragments and third electrophoresis (SD "hybridization of in-situ hybridization of DNA fragments ( Hm); a three-dimensional electrophoresis device (SD) has a central platform, and the electrophoretic fluid tank (NP ,,
  • N !, ⁇ 2 are negative electro-hydraulic tanks, and P, and P 2 are positive electro-hydraulic tanks; a stainless steel mesh is provided below the platform, and a negative electrophoretic liquid tank is provided below the stainless steel mesh (N 3 ), a gel sheet for two-dimensional electrophoresis of DNA fragments is placed on a stainless steel mesh, a hybrid gel film (Hm) is placed on the gel, and a stainless steel mesh (P 3 ) used as a positive electrode is set on the hybrid gel film ( Hm) above.
  • Another object of the present invention is to improve the non-natural defects in the prior art.
  • a method for screening natural procedures, based on and simulating natural procedures is established. That is, the gene activation region (methylation / demethylation) is modified. Methylation or enhancer insertion) or expression region (gene amplification), the method of switching its expression, or changing its expression level.
  • this method of controlling gene expression should be precise (specific and specific in situ control of target genes) and highly efficient (modifiers, morphogens and morphogens are small molecules, which are easy to synthesize or introduce; Modifications alter the DNA structure to allow stable passage of the modified phenotype in cells). Therefore, this method is particularly suitable for the screening of common fatal gene disease treatment drugs and the rapid breeding of excellent animal and plant varieties.
  • the present invention also relates to a method for modifying a gene to change its expression state, which comprises the following steps: In a first step, a method for isolating a phenotypic related gene according to the present invention is obtained from a plurality of patients suffering from the same disease.
  • Mutant fragments, or mutation fragments obtained from multiple different tissues with the same physiological phenotype detect gene mutations or modified fragments related to the disease or physiological phenotype; in two steps, use multiple gene mutations related to the same disease to detect Needle, or related with the same physiological phenotype Multiple genetic modification probes, screening the entire sequence of the target gene, and cloning and sequencing; determining the modified region, the promoter region and the expression region of the target gene; the third step is to determine the modification region and the promoter region by comparing with the synchronous gene Synthesize or clone these motifs with the recognition motifs of upstream genes in the expression region; in the fourth step, you can use the modification region or promoter region motif probes to directly hybridize with genomic DNA or cDNA to screen genes that express switch molecules You can also use these probes to hybridize with random or non-random (embryonic cDNA) phage peptide library (culture), bacterial protein library or yeast protein library, extract the positive clone D1NA as a probe
  • Figure 1 is a flowchart of a method for isolating phenotype-related genes.
  • Figure 2 is a schematic diagram of a three-dimensional electrophoresis apparatus for isolating phenotype-related genes.
  • Figure 3 is a schematic diagram of the structure of a hybrid gel membrane.
  • Figure 4 is a flow chart of the present invention's screening of switch molecules for treating common lethal diseases. among them
  • the human genome is very large and complex, including 6 X 10 9 nucleotide pairs and a high base rate of 100,000, and the goal of searching for small point mutations or point modifications from it cannot be achieved.
  • the human genome is a highly rigorous and ordered long chain molecule. All somatic cells in an individual divide from a fertilized egg cell. The highly sophisticated DNA replication mechanism allows them to have basically the same genome and its restriction map. Only a few regions have been mutated or transformed (modified or activated) You need to retrieve it.
  • the rigorous structure of the genome and the restriction fragment length polymorphism (RFLP) allow us to use homogeneous sequencing of two types of genomic DNA by mixing two cuts and two-dimensional electrophoresis.
  • restriction fragment in situ addition (RFASIS) method of the present invention changes the usual disordered hybridization between genomes into an ordered hybridization, and performs 10 9 hybridizations and searches on a gel at the same time. Therefore, the retrieval efficiency is greatly improved and the probability of errors is reduced. Thus, the ⁇ target for point mutation or point modification in the complex human genome was retrieved.
  • Isolating pathogenic genes As mentioned earlier, certain gene mutations or changes (modifications / activations) can cause a disease, and each gene has its own specific sequence. Therefore, as long as several hundred bp mutations, modifications or promoters between the diseased and healthy cell genomes are isolated, they can be used to screen out these disease-causing genes.
  • the specific method is: point mutation (base substitution or small deletion, insertion) makes a circular mismatch between the hybrid double strands, which can be separated by cleavage with a compound or enzyme, or repaired with affinity for the mismatch structure ⁇ Fixed and separated; point modification (methylation / demethylation of cytosine in the CpG sequence) makes a half-methylated site between the hybridized double strands.
  • the mutations or transformation fragments isolated in the above methods often mix a large number of fragments that are not related to disease.
  • the RFASIS method can be used to separate the truly pathogenic fragments from these unrelated fragments: 1. Select healthy cells with small differences from diseased cells as a control. For example, the formation of cancer cells passes through normal cells-benign tumors ⁇ carcinoma in situ-infiltrating cancer ⁇ metastatic cancer in multiple stages, and to isolate oncogenes of cancer in a certain stage. Cells in adjacent stages should be selected as control samples, which can make the number of mutant fragments Minimized.
  • the telomere gene is a gene that is active during DNA synthesis and may be present. The phase was started.
  • telomere gene expression spermatogonial cells with telomere gene expression and fibroblast Gi phase cells with no telomerase expression were selected for RFASIS. At this time, neither of the tissue-specific persuasive genes was expressed. . 2.
  • the same disease is caused by a mutation in the same disease-causing gene or the same genetic transformation.
  • the ectopic addition of mutant fragments obtained from different patients with the same disease can be used to isolate the pathogenic fragments on the same pathogenic gene between the two: first use the mutant fragments of a patient and the full-base group length restriction fragments Long enough to prevent the entire gene from being cut) hybridize and fix the large fragment on the hybrid; then use another patient's mutant fragment to hybridize with it, and fix the fragment on the hybrid this time, that is, the pathogenic fragment.
  • Another method is more convenient: after mixing the mutant fragments of multiple patients, using these fragments as primers, biotin nucleotides as substrates, and whole DNA as templates for PCR amplification, the same pathogenic diseases are similar
  • the fragment will be specifically amplified and fixed with biotin affinity protein. This kind of ectopic hybridization is less efficient than in situ hybridization, and there are more opportunities for cross-breeding.
  • mutant fragments are far less than the number of whole genome fragments, and they can be amplified by PCR to a large number, which can achieve sufficient hybridization efficiency; the crossover between family genes and allele homologous fragments is screened It is also a disease-related fragment, and crossover between repeats can be suppressed by pre-hybridization of known repeats.
  • Our experiments show that both ectopic hybridization and PCR amplification of adjacent pathogenic fragments can achieve satisfactory results.
  • in situ addition of transformed fragments obtained from different tissues with the same defect can also isolate pathogenic fragments that are homologous between the two: Mix the promoter fragments or modified fragments from different tissues with one-way electrophoresis and the original Position hybridization and then put Pathogenic fragments on the cross are fixed and isolated. 3.
  • a disease occurs when a mutation in a gene in a gene group that expresses a persuasive phenotype causes a defect in cells. All genes in this gene group can be called functional genes related to the disease, and the disease-causing genes are also one of the members.
  • the RFASIS method can be used to isolate various phenotype-related persuasive gene groups (see below for details). Then, by adding mutation fragments and persuasive fragments, the pathogenic fragments can be separated. In fact, the mutation, modification, promoter, and functional fragments associated with a disease are added to isolate the consensus sequence, which is the pathogenic gene fragment.
  • Isolating disease-suppressing genes For the treatment of genetic diseases, it may be more important to isolate disease-suppressing genes (genes that express normal cell functions disrupted by the disease). So far, there are still serious technical obstacles to repairing and replacing a mutant gene; in most cases, it is not difficult to find and activate a disease suppressor gene that can replace the mutant gene.
  • a gene group that expresses a certain kind of normal energization usually requires three switches to finally make the cell form this energizing phenotype. These three switches are the switch of gene modification region, promoter region and expression region.
  • the modified regions of the housekeeping gene, DNA synthesis gene, and cell division gene have been demethylated, and their promoter regions have been activated in cell G ( >,!, And 2 phases (in combination with transcription factors) and Expression, thereby forming the basic structure of cell life.
  • the modified regions of tissue-specific genes are demethylated, thereby forming the tissue phenotype of the cell.
  • genes including cell proliferation control genes, a variety of persuasive genes
  • the promoter region that can regulate and express genes, apoptotic genes, etc. will be in the cell proliferation cycle and, however, the expression of a functional gene group just generates two stationary energy protein information chains.
  • the gene expression region should have an upstream gene recognition region (regulatory subunit) and a downstream gene recognition region (catalytic subunit), and their cascaded (cascade) turn on and off for the cell to finally perform.
  • the RFAS1S method can be used to search for identified tissues, developmental phenotypes, or movements.
  • the differential fragments between the genomes of the normal cells and their precursor cells, or the homologous fragments between the genomes of cells with the same developmental phenotype in different tissues have also retrieved a certain permissive related gene group.
  • the modified fragment between hematopoietic stem cells / erythroid stem cells is the erythrocyte tissue phenotype-related gene;
  • the stem cell / mature cell starter fragment is the cell persuasive phenotype-related gene;
  • the mature cell / terminal cell starter fragment is the cell proliferation regulation Related genes;
  • the synchronized active fragments isolated by the above method can be hybridized with the random phage peptide library culture to isolate the same functional group fragments with the same modified region or promoter region; or sequencing research can persuade the gene group to modify the region or the promoter region. Sequence, this motif probe is used to screen synergistic group fragments in synchronization fragments.
  • the RFAS1S method can be used to search for gene groups related to various functional phenotypes, which is essential for gene switch therapy of aging diseases.
  • the gene modification region is located in the upstream promoter region of the gene expression sequence, and includes several CpG sequences.
  • the 5th carbon atom on the cytosine ring in the GpG dinucleotide is methylated ( 5 m CpG). It binds to a methylophilic sequence protein (MePI) and blocks the promoter region.
  • the start region includes the consensus sequences of TATA, CAAT and GC. These consensus sequences allow us to determine the approximate location of the modification and promoter regions.
  • members in the same functional group should have the same modified or promoter region. Therefore, by comparing the above regions of different genes in the same functional group, you can find the precise identification position of the switch (basic sequence).
  • switch molecules modify, activate, and activate specific gene populations to form the cell's tissue, developmental phenotype, and activity. These switch molecules are expressed in very low amounts. Or checking them is very difficult. But one thing is certain, that is, genes that express these switch molecules (including development genes that express modified molecules, regulatory genes that express starter molecules, and hormone genes that express activating molecules) Will be stored intact in genomic DNA. Moreover, their expression (RNA or protein) has specific affinity with the modified region, the promoter region or the expression region of the target gene being switched, and can be combined with it to form a stable structure. Therefore, we can use the identified switch region ( ⁇ ) motif probes to screen these genes that express switch molecules directly or through phage peptide libraries into genomic DNA.
  • RNA or protein RNA or protein. But there is evidence that proteins are more likely. In the nucleus, single-stranded DNA is usually wrapped with a single-stranded affinity protein, and the modified or promoter region is recognized as a double-stranded form. Therefore, they are unlikely to be recognized by single-stranded RNA. However, obviously, the possibility of RNA recognition cannot be completely ruled out. In addition, some genes are expressed in embryonic cells and no longer expressed in adult cells, such as the telomerase gene. It is not yet certain whether the non-expression of these genes is due to the fact that the genes have not been demethylated or that they have not been activated or activated. When searching for switch molecules, various possibilities should be considered: 1.
  • the gene has not been demethylated.
  • the cells ⁇ 2 ⁇ 2 / ⁇ 2 r 2 globin genes in the yolk are demethylated to express fetal globin. After that, these cells apoptotic, and the hepatocyte a 2 r 2 globin gene was demethylated to express fetal globin.
  • the ⁇ 2 r 2 / ⁇ 2 ⁇ 2 globin gene of bone marrow cells was demethylated to express adult globin, while fetal globin red blood cells accounted for 1%.
  • the embryo and fetuin are not expressed in the adult because the expressed cells have apoptotic, and these genes are not demethylated in the non-expressing cells.
  • many development hormone-expressing cells in human embryos, pineal glands, and thymus are activated with human birth, maturity, or the activation region is blocked. After a persuasion gene group is demethylated, It is expressed only when the source or endogenous transcription factor is activated.
  • the three temporal changes of cells from division, differentiation, and apoptosis are all in the way that the previous tense-expressed genes are turned off in a way that the promoter region is not activated or blocked.
  • the PAX-2 gene is activated in early renal stem cells, and is shut down in mature cells by the WT1 factor (expressed by the tumor suppressor gene WT1). In fact, somatic cell proliferation disorders are caused by genes that have not been activated or have been blocked. 3.
  • the gene is expressed, but the expressed puppet or protein is not activated.
  • the human pineal gland, thymus and gonadal all degenerate with aging, and as the hormones they secrete decrease and disappear, the target genes of these hormones are no longer activated.
  • the lack of thymus in the elderly prevents T-cells against self-antigens from apoptotic, and T-cells against foreign antigens do not mature, resulting in low cellular immunity.
  • gene expression is not shut down in any way.
  • Genes that express the keys (switch molecules) that turn them on are all methylated in adult cells. Demethylating this switch gene will also enable the target gene to be activated very simply and efficiently: Modification can be blocked with non-physiological molecules that are specifically screened from the random phage peptide library and can specifically bind to the genetic modification region Demethylation. To turn on the promoter region or activate the expression region, it is necessary to use physiological molecules with complex activation / activation activities. Second, the state of gene demethylation is preserved in cell passages, thus continuously providing switching molecules.
  • the gene By blocking the promoter or expression region with a polypeptide or antisense R1NA that is compatible with the promoter or expression region of the gene, the gene can be prevented from being activated or activated.
  • a polypeptide or antisense R1NA that is compatible with the promoter or expression region of the gene
  • Gene switching can treat and prevent most common fatal diseases.
  • Genetic transformation disease Diseases caused by physiological changes (switches) of genes include disorders of somatic cell proliferation, low tolerance of nerve cells to lack of gas, and lack of apoptotic gene activity in certain stem cells and naive cells, etc. Human beings are the most advanced form of life evolution today, but obviously there are still many unsatisfactory things. Loss of telomere Lives can progressively shorten telomeres and preferentially screen out those cells that divide too many times and accumulate more mutations. But it did trigger arteriosclerosis and cancer. It is necessary to modify the defects in life. For the treatment of this type of disease, all that is required is to turn off and turn on genes that have been turned on or off by mistake.
  • Genetic mutations can cause two types of disease. (1) Mutation inactivates the gene being expressed. Repairing and replacing mutated genes is difficult. However, many important persuasive genes are multiple copies, including alleles on homologous chromosomes and family genes arranged in clusters on the same chromosome. They generally have similar sequences and persuasion but different switching regions. Different family genes are activated during different individual developmental stages, and one copy of the allele is often dormant (called genomic imprinting and X chromosome inactivation). For example, the globin gene family includes eight copies, which are expressed in embryonic and adult stages, respectively.
  • any tissue-specific gene group retains dormant copies in other tissues.
  • dormant copies in other tissues can be activated to restore the body's lost counseling power. For example, when the pancreas is severely damaged, it can activate the insulin secretion-related gene group in bone marrow stem cells to restore this aspect.
  • tissue-enhancing and apoptotic genes in stem cells have not yet been activated. When these cells undergo proliferative disorders, these genes fail to be activated and cause cell canceration. Activation of these genes can effectively inhibit cancerization.
  • Mutations activate dormant genes. Uncontrolled proliferation, infinite proliferation, invasion, and metastasis (induced angiogenesis) of cancer cells are the characteristics of some dormant gene copies that are activated by mutations. Turning off one or two genes can effectively suppress cancer. The level of fat-promoting hormones in obese patients may be too high. Turning off the S-line of fat synthesis in the liver and fat cells can inhibit the accumulation of fat.
  • Gene switching does not treat all genetic diseases.
  • a healthy gene For some transmissional, single-copy genetic diseases, a healthy gene must be introduced instead of activating a dormant gene already in the body.
  • the efficiency of existing gene transduction techniques is too low (10 6 ). This may be due to the lack of expression of recombinant genes in somatic cells.
  • highly efficient gene-specific or non-specific homologous recombination does exist, for example, in meiotic cells.
  • RFASIS method can be used to search these homologs Recombinant genes and their promoters. When these genes are activated during gene transduction, efficient homologous recombination of a natural program can be achieved. Therefore, the switch gene method can be used to efficiently and accurately introduce a healthy gene into cells.
  • tissue phenotype modified with the target tissue-specific proliferation hormone, for example, ECGF when modifying vascular endothelial cells, red cells when With EPO, etc., the target tissue cells are modified and modified, and other tissue cells are at rest without modification.
  • target tissue-specific proliferation hormone for example, ECGF when modifying vascular endothelial cells, red cells when With EPO, etc.
  • the target gene modification region and the promoter region can be administered at the same time. For example, modify its proliferation-regulating genes to stop division and activate its apoptotic genes to promote its apoptosis. 3.
  • For non-dividing nerve and muscle cells you can start their division first, and then modify and modify them. There is evidence that nerve and muscle cells can also divide under certain pathological conditions. Their non-dividing is not that the modified region is closed, but that their cell proliferation regulating genes are not activated. Or it is not activated.
  • These promoter molecules or activating molecules are retrieved by RFASIS method, and they can be used to initiate cell division and then modify and modify them to treat this cell proliferation deficiency disease.
  • the method of switching genes according to natural physiological procedures can not only continue aging, prevent and treat various common somatic gene diseases, but also improve the efficiency of gene transduction methods.
  • Various rare genetic diseases can not only continue aging, prevent and treat various common somatic gene diseases, but also improve the efficiency of gene transduction methods.
  • Various rare genetic diseases can not only continue aging, prevent and treat various common somatic gene diseases, but also improve the efficiency of gene transduction methods.
  • Various rare genetic diseases can not only continue aging, prevent and treat various common somatic gene diseases, but also improve the efficiency of gene transduction methods.
  • the method for isolating a phenotype-related gene and a switch gene to regulate a gene expression state of the present invention may include:
  • the modification point of the separation point is fixed by modified ⁇ (methylation maintenance ⁇ , restriction ⁇ I) to fix and separate the DNA hybrid double-stranded hemimethylation site.
  • the control fragment Before the sample and the control fragment are mixed and electrophoresed, the control fragment is methylated or biotinylated. After they are fully hybridized, the restriction of recognition of the methylation site is used. The mutant sample hybridized to the control sample is cut and separated; or the control sample and the hybridized fragment are fixed with a biotin affinity protein, and the unhybridized sample mutant fragment is separated. Subtraction can also be used to separate point-modified fragments: after mixing two-dimensional electrophoresis of the sample and the control fragment, the unmethylated fragment is cut with a restriction enzyme that cuts unmethylated CpG, and electrophoresed vertically (vertical to the gel surface) to another film on. Subtract the point modified fragments of the sample.
  • the subtraction separates the enable / enable segments.
  • CDNA was used as the sample and control.
  • the method is the same as above, and mRNA ectopic hybridization can also be used: firstly, the control mRNA is hybridized with restriction fragments of genomic DNA, and then hybridized with biotinylated PolyT, and then hybridized with mRNA with biotin affinity protein. The intersecting restriction fragments are separated. Then use the sample mR A to hybridize with the remaining restriction fragments, and use biotinylated poly T to fix the fragment hybridized to the mRNA, which is the activation / encouragement fragment.
  • mutant and transformed fragments isolated by the above addition and subtraction methods can be amplified into probes by PCR using double linkers.
  • One of the modified or activated fragments obtained from different cells with the same physiological or pathological phenotype is labeled with biotin, and after hybridization in one-dimensional electrophoresis in situ hybridization, a fragment is first hybridized with a biotin affinity protein The fragment is immobilized and another fragment that does not hybridize elutes from the gel. Then the denaturation process separates the fragments on the hybridization, that is, the phenotypic related fragments.
  • Modified or promoter motif probes can be directly hybridized with genomic DNA or cDNA to screen genes that express switch molecules; these probes can also be used with random bacterial protein libraries (embryo cDNA); random yeast protein libraries ( Blastocyst cDNA) (culture) hybridization, extract the DNA of the positive clone as a probe, and then screen it in the DNA or cDNA. (See Figure 4 for details)
  • the expression region motif can be introduced into a phage to express the corresponding polypeptide. Then use this as a probe to hybridize with a random bacterial protein library (embryo cDNA); a random yeast protein library (embryo disc cDNA), extract the DNA of the positive clone as a probe, and then screen it in the DNA or cDNA.
  • a random bacterial protein library embryo cDNA
  • a random yeast protein library embryo disc cDNA
  • DNA homology between two genomic sequencing can be both the DNA hybridization complex from 107 to 10 kinds or two or less, the degree of resolution can be reduced to a single restriction fragments of the two genomes difference.
  • Restriction fragment in-situ addition and subtraction can be used to retrieve differences between genomes caused by any form of genetic activity (mutation, modification, activation, and activation). 3. Mixed genomic DNA between sample and control. Two-dimensional electrophoresis and crossover / self-crossing fragment subtraction can avoid false negative or false positive results caused by most misalignments and crossovers. 4. Differential fragments of different genomes with the same physiological or pathological phenotype. 5. The switch region motif of a known physiological or pathological phenotype-related gene can be used to retrieve the natural and physiological molecules that switch the gene expression and the upstream genes that express the switch molecule. 6. Inactivating disease-causing genes and activating disease-suppressor genes through switch molecules can effectively control and inhibit most common fatal diseases.
  • the hybrid gel membrane (Hm) in the device of the present invention includes a square nylon membrane (Nm) or a nitrocellulose membrane, and the membrane pore diameter is 0. ⁇ , and a nylon membrane (Nm) is attached with a thin layer of 1-2 mm.
  • the acrylamide hollow gel has a concentration of 0.8%, and the pores of the hollow cells (Cell) have a size of 10 ⁇ m to 100 ⁇ m, and the cells contain liquid or low concentration Hydrolink gel or agarose.
  • the method of the invention can be used to screen related genes and therapeutic drugs for common fatal diseases. The following are examples of active factors that can be screened by the method of the present invention.
  • TAF Telomerase activating factor
  • NPAF Nerve / muscle cell proliferation activating factor
  • GSF Growth hormone inhibitory factor
  • Apoptosis activating factor (CAAF)
  • EGAF Embryo globin activating factor
  • CISF Cancer Invasion Inhibitory Factor
  • CMSF Cancer metastatic inhibitory factor
  • HRAF Homologous recombination activating factor
  • Enhancer insertion activation factor (EIAF)
  • EPOAF Erythropoietin activating factor
  • HPOAF Hepatopoietin activating factor
  • IFN Insulin activating factor
  • Atrial natriuretic factor (ANFAF)
  • Tissue-type fibrinolytic activator t-PA activating factor (t-PAAF);
  • HRF Hypothalamic release hormone activating factor
  • the DNA extraction method is the same as in Example 1.
  • RFAIS restriction fragment in situ subtraction
  • Example 4 Clone the isolated target fragment into a probe:
  • the sample and control DNA fragments were labeled with radioisotopes and biotin, respectively.
  • the two were mixed at 1: 100 ratio in Ao gel two-dimensional electrophoresis (TE).
  • TE Ao gel two-dimensional electrophoresis
  • all the DNA fragments were transferred into the hybrid gel membrane 1 containing biotin avidin, and the biotin labeled Compare this fragment.
  • Neutral fluid treatment then hybridizes the non-mutated fragment to the control fragment. Transfer the unhybridized mutant fragments to another hybrid membrane (SH).
  • X-ray autoradiography (Aa) was performed on the B, membrane to show the position of the mutant fragment, and it was removed and further amplified as a probe. This method is suitable for isolating mutant fragments smaller than 100bp.
  • This method is similar to Example 5, except that the former uses the restriction of the CpG sequence that does not cleave demethylation mutations, and performs the second 6: cut, and the latter uses cleaved CpG without cleavage.
  • the base of the CpG sequence is ⁇ , and the mutant sample of the sample has one less cut point than the control fragment. The displacement between the two can be separated by hybridization subtraction.
  • sample and control DNA fragment (RF) at 3, respectively. Isotope and biotin. The two were mixed in Ao gel and subjected to unidirectional electrophoresis. After the alkaline solution was denatured, the DNA was transferred to another gelatin membrane containing biotin avidin. The control fragment was incubated and fixed, and then refolded with neutral solution. The non-rearranged sample fragments are hybridized and fixed on the membrane, the rearranged mutant fragments are transferred to the membrane (SH), and then the point mutation fragments in the Ai film are cut with chemicals or osmium, and the free point mutations are cut. The fragment was transferred to the film B 2 (AH 2 ). Finally, the film A 2 from which the rearranged and point-mutated fragments were removed was subjected to X-ray autoradiography (Aa). The display position was the same fragment position of the blood relative. Further amplified as probes.
  • the sample and control DNA fragments (RF) were treated with demethylated and methylated enzymes, respectively, and the two were mixed in Ao gel at a ratio of 1: 100 by two-dimensional electrophoresis and in situ hybridization. Then use the restriction S that does not cut the methylation sequence to cut the self-double-stranded samples (that is, rearranged mutant fragments) of the demethylated samples, and finally transfer these cut mutant fragments to the membrane B 2 (AH, ). Neither the methylated control fragment nor the hemimethylated hybrid fragment was cut short, and they fell behind in the gel film A 2 during electrophoresis. Then the mutant fragment in the membrane B 2 was further amplified as a probe.
  • Example 10 Isolation of phenotypic related mutations or modified fragments using restriction fragment comparison subtraction.
  • the restriction standard was used to cut human standard DNA into an average length of 100 kb, and the gel was subjected to pulsed field electrophoresis into the gel. The DNA fragment was fixed in the gel at low temperature or dehydration.
  • the modified fragments obtained in cells are labeled with different luciferin, and then they are hybridized to the above-mentioned fixed DNA.
  • the unhybridized probes are washed away, and the two-color spots are searched with an automatic fluorescence scanner.
  • Disease-related or physiological phenotype-related genetic loci Take out the DNA at this site, mark the end of the probe with biotin or digoxin, and fix it. After the probe and the large DNA-related fragments are fully hybridized, remove the unrelated large fragments by electrophoresis. The remaining fragments may contain genes. Full sequence.
  • Example 10 Operate as in Example 10 above. If no two-color dots are screened, you can choose different restrictions, or cut the DNA into longer fragments for screening. If too many two-color dots are screened, there may be overlaps of single-color dots. Separate them by two-dimensional electrophoresis, you can gradually search for longer fragments to ensure that the complete gene sequence is included; you can also use more probes screened from patients to ensure that the entire gene sequence is included Sequence. Finally, through Southern blot analysis of multiple probes, it was determined that there were several target genes and the size of the target genes.
  • Example 12 Screening of morphins using methylation / demethylation sequence probes in the promoter region of target genes.
  • Many genes for example, the telomere gene
  • This methylation sequence probe can be used to directly hybridize with protein or RNA electrophoresis gels in the nucleus, thus showing the moldin that causes methylation / demethylation of the target gene.
  • Drugs can be designed based on the moldin (for example, it can be designed Drugs that activate the telomere gene in vascular endothelial cells to treat arteriosclerosis; Drugs that inactivate the telomere gene in tumor cells to treat cancer) Change the target gene expression status.
  • genes such as genes that control embryonic cell-independent proliferation
  • a signal such as morphogen
  • morphogen can be screened according to the method of Example 12 to directly design a drug (for example, activate Genes that control independent proliferation in tumor cells) can also be used to further investigate upstream genes based on morphogens and to design drugs that change the expression of upstream genes (formin).
  • a small number of genes are highly expressed by enhancers or modified expression regions.
  • a gene for example, human serum albumin gene in pig cells
  • the albumin gene was further modified to increase r3 ⁇ 4 expression.

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Abstract

L'invention concerne un procédé et un dispositif servant à disperser un fragment de restriction, au moyen d'une digestion enzymatique et d'une électrophorèse à deux directions. Ce procédé consiste à isoler le fragment mutant apparenté au phénotype, par hybridation in situ à l'aide de la méthode du plus-moins, à cribler directement toute la séquence du gène par une technique de marche le long d'un chromosome, au moyen dudit fragment utilisé comme sonde et servant à déterminer la région modifiée, la région promoteur et le motif de la région d'expression, puis à cribler le gène aux fins d'expression d'une transition moléculaire avec la sonde conçue à l'aide du dernier fragment, et à réguler l'expression génique à l'aide de la translation moléculaire exprimée par ledit gène ou le réactif conçu à partir de celui-ci. L'invention est notamment appropriée à un criblage de médicaments thérapeutiques destinés à soigner la plupart des maladies mortelles communes et à promouvoir ou accroître l'expression de ce gène à caractère supérieur chez des animaux et plantes à usage industriel.
PCT/CN1997/000136 1996-11-25 1997-11-25 Procede d'isolement d'un gene apparente a un phenotype, region de transition ainsi que transition moleculaire de ce gene WO1998023736A1 (fr)

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AU51151/98A AU5115198A (en) 1996-11-25 1997-11-25 A method for isolating phenotype related gene and their switch region as well as their switch molecular

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CN96119585A CN1057338C (zh) 1996-11-25 1996-11-25 分离表型相关基因的方法及其装置
CN96119585.1 1996-11-25
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CN 97109139 CN1167149A (zh) 1997-06-16 1997-06-16 修饰基因以改变其表达状态的方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038515A1 (fr) * 1999-11-19 2001-05-31 Xiaozhuang Bian Procede d'isolation de molecules a changement de phenotype et batteries de genes relatives a un phenotype

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009334A1 (fr) * 1987-05-26 1988-12-01 Calgene, Inc. Facteurs de transcription specifique au fruits

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988009334A1 (fr) * 1987-05-26 1988-12-01 Calgene, Inc. Facteurs de transcription specifique au fruits

Non-Patent Citations (3)

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Title
EUROPEAN JOURNAL OF IMMUNOLOGY, 27(11), 1997, HANSEN J.D., STRASSBURGER P., DU PASQUIER L., "Conservation of a Master Hematopoietic Switch Gene During Vertebrate Evolution: Isolation and Characterization of Ikaros and Teleost and Amphibian Species", pages 3049-3058. *
KOSMOS (WARSAW), (1987), 36(2), PLUCIENNIC ZAK, ANDRZEJ, "Possibilities of Genetic Engineering in the Production of Drugs and Vaccines", pages 177-88. *
PROC. NATL. ACAD. SCI. U.S.A., (1997), 94, 22, DRYJA T.P., "Gene-Base Approach to Human Gene-Phenotype Correlation; Mutation Detection and Human Genome Mapping", pages 12117-21. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038515A1 (fr) * 1999-11-19 2001-05-31 Xiaozhuang Bian Procede d'isolation de molecules a changement de phenotype et batteries de genes relatives a un phenotype

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