159results about How to "Stable amplification" patented technology

The invention relates to a chromosome specific marker of elytrigia elongata in wheat background and a use thereof. Primers are designed according plant EST sequence information, a wheat-elytrigia elongate addition line is amplified by a PCR technology to form a specific DNA fragment of elytrigia elongate, the specific DNA fragment is sequenced, novel primers are designed according to a sequence nonhomologous with wheat, and the elytrigia elongata chromosome specific molecule marker is obtained. The chromosome specific marker can be used for translocation line identification in chromosome fragment transfer from elytrigia elongata to wheat, can be used for gibberellic disease-resistant gene linkage analysis, can be used as a specific molecule marker for identification of elytrigia elongata chromosome and can be used for wheat gibberellic disease resistance and stress resistance breeding. The chromosome specific marker solves the problem that through a development technology of molecule markers such as RAPD, AFLP and SSR, specific markers of a part of chromosomes are obtained, the developed elytrigia elongata chromosome specific markers are less and cannot satisfy actual requirements on wheat resistance breeding and chromosome singularity and stability are non-ideal.

The invention belongs to the field of crop genetic breeding. Particularly, a primer is designed according to repeated sequence information of a plant; an additional wheat-thinopyrum elongatum system is amplified by utilizing a PCR (polymerase chain reaction) technology to obtain and sequence a specific DNA (deoxyribonucleic acid) segment of thinopyrum elongatum; the primer is designed again according to the sequence without homology to that of the wheat so as to obtain the specific molecule mark of thinopyrum elongatum chromosome, wherein the molecule mark is one of eight pairs of primers provided by the invention. The marks can be used for translocation line identification in the process of transferring chromosome segments from thinopyrum elongatum to wheat, can be used for chain analysis of a gibberellic disease resistant gene, and can be used as the specific molecule marks to identify thinopyrum elongatum chromain for gibberellic disease resistance and stress resistant breeding of wheat.

The invention belongs to the field of crop genetic improvement, comprising the following steps of: sequencing the specific segments of common wheat and a common wheat-elytrigia elongata addition line to obtain a mass of common wheat and elytrigia elongata sequences, carrying out comparative analysis on the sequence data by computer software to obtain a mass of elytrigia elongata 1E chromosome specific segmental sequences, randomly selecting 8 sequences, and successfully developing 5 elytrigia elongata 1E chromosome specific molecular markers such as one of SED ID NOs.1, 4, 7, 10 and 13. The markers not only can be used for detecting the elytrigia elongata 1E chromosome, but also can be used for the assistant selection of the molecular markers in the wheat resistance breeding. Furthermore, the SLAF-seq technology-based successfully developed elytrigia elongata chromosome specific molecular markers can provide an important reference for the molecular marker development technologies of the other species, and provide an important application basis for the molecular breeding, the system evolution and the germplasm resource identification.

The invention discloses a method for stably amplifying high-stability and high-cytotoxicity NK cells in vivo. The method for amplifying the NK cells in vivo comprises the following steps: (1) separating single mononuclear cells; (2) carrying out immune sorting to remove CD3<+>T cells; (3) adding a serum free medium containing cell factors (with the final concentrations of 10 to 500ng/ml IL-15, 10to 500ng/ml IL-12, 10 to 500ng/ml IL-21, 10 to 500ng/ml IL-18 and 500 to 2000U/ml IL-2) needed by NK cell amplification; (4) wholly replacing the NK cells with liquid in the 2nd to 5th days; (5) harvesting the NK cells. The method disclosed by the invention can be used for stably obtaining a lot of the high-stability and high-cytotoxicity NK cells and the NK cells can be used for immunotherapy oftumor cells.

The invention discloses an EST-SSR core primer group for identifying the variety of Chinese wolfberry and an identification method and an application thereof. Through screening of EST sequences of lycium ruthenicum and Ningxia wolfberry, a large number of SSR molecular markers are developed, and an SSR marker-meeting fluorescence detection technology system including 10 markers is established and is used for identification of the Chinese wolfberry variety and species. The 10 markers have the advantages of good polymorphism, stable amplification, clear amplification results and good repeatability, can be used for large-scale detection of authenticity of Chinese wolfberry seedlings and the purity of the seedlings, can provide a technical basis for standardization of seedling markets, and are conducive to development and use of the traditional Chinese medicine Chinese wolfberry.

The invention discloses a molecular marker Xwmc388-205 closely linked with QTL resistant to wheat crown rot and application of the molecular marker Xwmc388-205. The molecular marker is a DNA segment with the size of 205 bp, wherein the DNA segment is obtained in the mode that the a wheat DNA is adopted as a template, PCR amplification is conducted through primer pairs of with nucleotide sequencesshown as SEQ No.1 and SEQ No.2, and , and then electrophoretic separation is conducted through 12% polyacrylamide gel of 12%. The molecular marker Xwmc388-205 can conduct forecasting and screening indoors by detecting resistance of the molecular marker to wheat crown rot, susceptible plants are weeded out, and thus the breeding efficiency is improved.

The invention discloses an SSR (Simple Sequence repeats) primer group based on Fraxinus Velutina Torr. transcriptome sequencing information development and an application of the primer group in germplasm identification, belonging to the technical field of molecular biology. The SSR primer group comprises 42 pairs of primers, wherein the nucleotide sequences of the first pair to the forty-second pair of primers are shown in SEQ ID No.1 to SEQ ID No.84. Experiments verify that the SSR primer group disclosed by the invention has the advantages of rich polymorphism, good repeatability, clear electrophoretic band and the like. The SSR primer group can be effectively used for researches on germplasm identification, DNA fingerprint establishment and the like. A germplasm identification method is sensitive and reliable, can be used for rapidly and accurately realizing and finishing distinguishment and identification of Fraxinus chinensis germplasm, and has important significances in identification and intellectual property protection of Fraxinus chinensis germplasm resources and genetic breeding of genetic breeding molecules.

The present invention relates to a method for culturing natural killer cells (NK cells) applied to immune therapy and, more particularly, to a medium addition kin for culturing NK cells with which lymphocytes derived from human peripheral blood can be cultured to effectively amplify and activate NK cells, which have an outstanding therapeutic effect on malignant tumors, while remarkably increasing the share of NK cells, and an NK cell culturing method using the kit.

The invention provides an SSR molecular marker used for indentifying germplasm resources of polygonatum Mill., and applications thereof, and belongs to the technical field of germplasm resource identification assisted by plant molecular markers. The SSR molecular marker is one or a plurality of molecular markers selected from the following seven SSR molecular markers. The seven SSR molecular markers are obtained by amplification through primers shown as SEQ ID NO.1-14. The provided SSR molecular marker can differentiate different germplasm resources of polygonatum Mill., with operation being simple, repeatability being good and accuracy being high. The SSR molecular marker can effectively overcome a defect that different germplasm resources of polygonatum Mill. cannot be accurately differentiated by utilizing phenotype identification methods, and is of great significance for polygonatum Mill. germplasm resource identification and molecular maker assisted selection.

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.

The invention discloses a SSR core primer set based on Pennisetum purpureum Schum transcriptome sequence development and its application. The SSR core primer set comprises 50 pairs of primers and the primers have nucleotide sequences shown in SEQUENCE ID NO. 1 to 100 in the sequence table. The invention also relates to a use of the SSR core primer set in Pennisetum purpureum Schum genetic diversity analysis and Pennisetum purpureum Schum germplasm genetic genealogy analysis. The 50 pairs of the SSR primers are derived from Pennisetum purpureum Schum spire transcriptome sequences. Compared with the existing Chinese pennisetum common SSR primers, the SSR core primer set has the advantages of high polymorphism, stable amplification, good repeatability and statistics convenience. The primers are successfully applied to Pennisetum purpureum Schum genetic diversity analysis and Pennisetum purpureum Schum germplasm genetic genealogy analysis, increase the amount of SSR primers used by Pennisetum purpureum Schum, can be effectively used for Pennisetum purpureum Schum genetic diversity analysis, identification of variety, fingerprinting construction and molecular marker-assisted breeding, and other Chinese pennisetum related sturdy. The SSR core primer set is suitable for promotion and application in the field of biotechnology.

The invention discloses a molecular marker Xgwm37-140 closely linked to the wheat stalk rot resistance QTL and application thereof. The molecular marker is a 140bp DNA fragment obtained by using wheatDNA as a template, performing PCR amplification through PCR primers with nucleotide sequence as shown in SEQ No.1 and SEQ No.2 and separating through 12% polyacrylamide gel electrophoresis. Accordingto the molecular marker Xgwm37-140, the wheat stalk rot resistance can be predicted and screened by detecting the molecular marker indoors, so as to eliminate infected plants and improve the breedingefficiency.

The invention relates to the field of genetic disease gene detection, in particular to a PCR primer group for amplifying a PKD1 gene. The PCR primer group comprises one or more pairs in a first primerpair, a second primer pair, a third primer pair and a fourth primer pair. According to the primer group disclosed by the invention, the length of an amplicon covering an exon No.1 is increased, stable amplification of an exon No.1 region is successfully realized, and an amplification product covers all exon regions and most of intron regions of PKD1; a PKD1 gene sequence is specifically obtained,and a pseudogene sequence of PKD1 cannot be completely amplified; and under the condition that pseudogene interference is completely eliminated, the variation of the PKD1 gene, including known variation and unknown variation, can be detected more accurately.

The invention relates to molecular markers linked to a ZYMV (zucchini yellow mosaic virus) dominant disease-resistant gene ZYMV-1 and application of the molecular markers. The genetic distances between the molecular markers ZY-138 and ZY-157 and the disease-resistant gene are 0.4 cM and 2.6 cM respectively. The molecular markers ZY-138 and ZY-157 are subjected to PCR amplification with primers; and whether a resistance character transformed progeny plant has the disease-resistant gene ZYMV-1 or not can be accurately detected through the products of amplification. The molecular markers have the benefits as follows: the efficiency of breeding for disease resistance is remarkably improved, the cycle of breeding is provided, and a guarantee is provided for selection of ZYMV disease-resistant varieties; and the molecular markers ZY-138 and ZY-157 have the advantages of reliability, stability, simplicity in operation, high repeatability and the like and achieve very high utilization values in distinguishing resistant/susceptible varieties and resistant individual plants.

The invention discloses a method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro, and particularly relates to a method for stably amplifying a large number of the high-purity and high-cytotoxic-activity NK cells in vitro by autologous or allogeneic peripheral blood mononuclear cells, which comprises the following steps: (1) separating mononuclear cells; (2) removingCD3<+> T lymphocytes in the mononuclear cells through immune sorting; (3) adding a serum-free cell culture medium for NK cell culture and cell factors IL-15, IL-12, IL-21, IL-2 and OK432, carrying out in-vitro culture for 2-5 days, and completely changing a solution; and (4) changing the NK cell solution; and (5) harvesting the NK cells. According to the invention, a large number of the high-purity and high-cell-activity NK cells are stably amplified under the in-vitro culture condition, meanwhile, the number of T cells is reduced, and the requirement for clinical application is satisfied.