38 results about "Suppression subtractive hybridization" patented technology

This invention relates to provide the genes for diagnosing colorectal cancer, the gene sequences searching comprise the steps of: (1) deriving epithelium cells from normal intestines, polypus of intestines and colorectal cancer tissue; (2) collecting genes with highly differential gene expression by Suppression Subtractive Hybridization (SSH), and building library; (3) deriving colonies with relatively high signal intensities from cancer tissue; (4) collecting more clinically cancer tissues by Northern Hybridization, real-time Polymerase Chain Reaction (PCR) combined with analysis of bioinformation to affirm variation between differential gene expression; and (5) selecting the most suitable genes from said library, and using the gene sequence as reagent provides the effects of early diagnosis, specificity, highly sensitivity and safety.

The invention provides soybean purple acid phosphatase GmPAP36 and an encoding gene and application thereof. A low-phosphorous induction soybean root system cDNA subtractive library is established based on the suppression subtractive hybridization technique, low-phosphorous stress inducible expression acid phosphatase candidate EST is selected from the subtractive library, and on this basis, an acid phosphatase candidate gene sequence is cloned and the expression difference of a candidate gene under the phytate phosphorus processing condition is analyzed with the real-time quantitative PCR technique; a gene prokaryotic expression vector is established to achieve gene prokaryotic expression; meanwhile, a gene eukaryotic expression vector is established, arabidopsis and soybean conversion is conducted to obtain transgenosis positive plants, the biological function of the gene GmPAP36 is analyzed, and functional genes are provided for efficient transgenosis breeding of plant phosphorus.

Methods of identifying the presence of differentially expressed transcripts associated with the presence of at least one circulating tumor cell in a biological sample of a cancer patient involves the use of suppression subtractive hybridization to identify differentially expressed transcripts present in biological samples of cancer patients not present in healthy patients, as well as differentially expressed transcripts present in biological samples of healthy patients and not present in the cancer patients. Utilizing these methods, a gene expression profile associated with a specific cancer phenotype can be constructed.

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 encode an intracellular protein that is expressed in prostate epithelial cells in a hormone dependent manner. Encoded proteins SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 have predominantly perinuclear, nuclear and predominantly nuclear location localization within a cell, respectively. In contemplated methods of detecting a neoplastic cell in a system, a predetermined amount of at least one of SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO: 6, or at least one of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is correlated with the presence of a neoplastic cell and detected within the system employing specific binding of a labeled probe. In a method of identifying differentially expressed genes, a tissue specific array of cDNA prepared by suppression subtractive hybridization is arranged on a solid phase. Two nucleic acid preparations are individually hybridized with the array, wherein the first and second nucleic acid preparations are prepared from treated and untreated target tissue. A comparison of the hybridization patterns reveals differentially expressed genes.

The present invention relates to cordycepin biosynthesis relevant gene and its application. Seven cordycepin biosynthesis relevant genes are cloned by means of suppression and subduction hybridizing (SSH) technology. They are named separately as chcs1, chcs2, chcs3, chcs4, chcs5, chcs6 and chcs7 and have cDNA sequence lengths of 760bp, 774bp, 713bp, 823bp, 746bp, 763bp and 890bp separately. Through constituting high efficiency plant expression converting vector containing the said genes, it is possible to increase the copy quantity of biosynthesis gene inside thallus and to raise the content of cordycepin aweto expresses for meeting the requirement in developing medicine.

This invention relates to a novel Gossypium hirsutum gene containing 26S rRNA sequence. The gene (GH18Rorf392) is obtained by: cloning EST sequence of cotton restorer through suppression subtractive hybridization method, designing primers according to the EST sequence, and performing 5'- and 3'-RACE to obtain full-length cDNA sequence. The gene comprises 26S rRNA sequence and 3'-end and a novel sequence at 5'-end. The gene is derived from Gossypium hirsutum restorer Y18R, and codes a protein of 392 amino acids. The gene can be used for studying fertility of cotton.

Loline alkaloids (LA), which are 1-aminopyrrolizidines with an oxygen bridge, are produced by Epichloë (anamorph=Neotyphodium) species, endophytes of grasses. LA are insecticidal, thus helping protect host plants from insect herbivory. Suppression subtractive hybridization PCR was used to isolate transcripts up-regulated during loline alkaloid production in cultures of Neotyphodium uncinatum. Subtracted cDNAs were cloned, and a λ-phage cDNA library from an LA-expressing N. uncinatum culture was screened with subtracted cDNA. In BLAST searches, several cDNAs identified had sequence similarities to aspartate kinases, and another with O-acetylhomoserine-(thiol)lyase. Differential expression of these two genes in LA-producing cultures of N. uncinatum was confirmed, and in a survey of 23 isolates from 21 Neotyphodium and Epichloë species these two genes strictly correlated with LA production. Two nucleic acid molecules encoding two loline alkaloid gene clusters have been identified.

A plant nutrition storage protein (GmVSPB) gene belongs to the technical field of genetic engineering and is characterized in that soybean Dongnong50 cotyledonary node is used as a material in suppression subtractive hybridization of differential gene when induced by cytokinin 6-BA so as to obtain a regeneration-associated gene and carry out gene cloning; a plant is transformed by agrobacterium-mediated transformation, changes of the transgenic plant are observed, gene functions are preliminarily analyzed by a quantitative RT-PCR method, and the influence of the cytokinin on regeneration is researched; starting from the influence of cotyledonary node organogenesis regeneration binding hormone on expression of soybean regeneration pathway associated transcription factor gene, effects of regenerating gene in a regeneration system are analyzed, and a soybean plant nutrition storage protein (GmVSPB) gene (Seq ID No:1) is found out.

The invention discloses a plant high-pH saline-alkaline tolerance gene SucHP and researches the expression of a high-pH saline and alkaline stress induction gene in a common seepweed herb seedling period by using suppression subtractive hybridization and high-density lattice film technology. The plant high-pH saline-alkaline tolerance gene SucHP can be prepared by the following steps of: firstly separating a gene of a coding tonoplast proton pump from common seepweed herbs; constructing a sense expression vector of the gene; and converting Arabidopsis. A transgenic plant has high high-pH saline-alkaline tolerance and can normally grow under the conditions of 100mM NaHCO3+200mM NaCl and 8.5 of pH. The plant high-pH saline-alkaline tolerance gene SucHP can be used for plant genetic transformation and increases plant saline and alkaline resistance.

The invention discloses a cDNA chip for detecting the resistance level of Bactrocera dorsalis against trichlorfon and beta-cypermethrin, a production method and application thereof. The present invention utilizes the forward suppressive subtractive hybridization (suppression subtractive hybridization, SSH) method to respectively obtain different resistant strains of Bacteralis dorsalis drug resistance-specific up-regulated expression gene fragments, and then PCR amplifies each drug resistance-related specific expression gene, purifies and recovers 118 specific gene fragments related to resistance to trichlorfon of Bactrocera dorsalis and 103 specific gene fragments related to resistance to beta-cypermethrin of Bactrocera dorsalis were obtained. Using the above-mentioned specific resistance target gene fragments as DNA probes, a cDNA chip for the detection of Bacteralis dorsalis resistance to trichlorfon and beta-cypermethrin was constructed. The cDNA chip of the invention can simply and effectively realize the real-time monitoring of the drug resistance level of Bactrocera dorsalis.

The invention provides a method for homogenization subtractive hybridization of full length cDNA. The method comprises the following steps in sequence: (1) synthesizing a first chain cDNA; (2) synthesizing a double chain cDNA; (3) selective connection and primer extension; (4) subtractive hybridization; (5) PCR augmentation of cDNA of differential expression; wherein, the PCR products of the subtractive hybridization can be directly cloned to a vector so as to construct cDNA libraries, where differentially expressed genes are concentrated; the libraries can be utilized to analyze gene expression, prepare gene chips and be marked as probe which selects the differentially expressed genes from the existing libraries; in addition, if the subtractive efficiency is not high enough, the subtractive hybridization can repeat for the PCR products of subtractive hybridization to improve the homogenization and the subtractive efficiency. The method for homogenization subtractive hybridization of the full length cDNA (FNSH) creates the beneficial effects of high subtractive efficiency of differential genes, high efficiency of enriching differential expressed genes, high homogenization and gainof the full length cDNA.

The invention provides a method for screening natural antisense transcripts related to the human tumor. The method comprises the following steps: extracting cancer cell / normal cell total RNA (ribonucleic acid); performing DNase I enzyme cutting and RNase I enzyme cutting; performing reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), performing double-chain synthesis, and adding linkers; performing suppression subtractive hybridization, cloning the polymerase chain reaction (PCR) product to establish a library, and performing sequencing analysis; and further performing experimental verification to candidate molecules and the like. After the method is applied and verified in normal cells and tumor cells, the method is proven to screen the natural antisense transcripts related to human tumor systematacially and rapidly.

The invention provides soybean purple acid phosphatase GmPAP36 and an encoding gene and application thereof. A low-phosphorous induction soybean root system cDNA subtractive library is established based on the suppression subtractive hybridization technique, low-phosphorous stress inducible expression acid phosphatase candidate EST is selected from the subtractive library, and on this basis, an acid phosphatase candidate gene sequence is cloned and the expression difference of a candidate gene under the phytate phosphorus processing condition is analyzed with the real-time quantitative PCR technique; a gene prokaryotic expression vector is established to achieve gene prokaryotic expression; meanwhile, a gene eukaryotic expression vector is established, arabidopsis and soybean conversion is conducted to obtain transgenosis positive plants, the biological function of the gene GmPAP36 is analyzed, and functional genes are provided for efficient transgenosis breeding of plant phosphorus.

The invention belongs to the technical field of plant gene engineering, and in particular relates to improvement of DON (deoxynivalenol) resistance and FHB (fusarium head blight) resistance of arabidopsis by utilizing wheat genes. A gene TaMetRS which is capable of improving DON resistance and gibberellic disease resistance of arabidopsis can be obtained through separation. By utilizing a method of screening a wheat suspension cell suppression subtractive hybridization cDNA (complementary deoxyribonucleic acid) library and full length of RACE (rapid-amplification of complementary deoxyribonucleic acid ends) clone genes, a gene TaMetRS which is not induced by DON can be separated from a wheat variety. By utiizing an agrobacterium tumefaciens mediated genetic transformation method, the gene is excessively expressed in arabidopsis, the resistance of the transgenic arabidopsis to the DON and the gibberellic diseases can be improved, and tests demonstrate novel application of the gene with a plant gibberellic disease resistance function. The nucleotide sequence of the gene is a sequence corresponding to a base at a site 1-2186 in SEQID NO:1, and 596 amino acids are encoded. The protein sequence encoded by the gene is as shown in SEQ ID NO:2.

The invention belongs to the field of crop genetic breeding, in particular 36 Lophopyrum elongatum genome-specific molecular markers. The 36 genome-specific molecular markers are obtained by the following steps: according to a nucleotide sequence of 18srRNA gene disclosed by National Center of Biotechnology Information (NCBI), designing primers in the sequence between two RsaI loci, removing homologous sequences between two genome DNA by using a suppression subtractive hybridization technology, enriching differential sequences, obtaining Lophopyrum elongatum specific DNA segments, sequencing the DNA segments, and redesigning primers according to sequences without homology with wheat. The markers can be used for identifying translocation lines in the process of transferring chromosome segments from Lophopyrum elongatum to wheat, can be used for linkage analysis of scab-resistant gene, and can serve as specific molecular markers for identifying chromatin of the Lophopyrum elongatum to be applied to scab-resistant and stress-resistant breeding of wheat.