73 results about "Southern blot" patented technology

This invention relates to a method of incorporating an exo-sample nucleotide into the amplified product strands resulting from a nucleic acid amplification process. Once the product strands have been obtained and analyzed (e.g., by hybridization, Southern blot, etc.), the exo-sample strands can be selectively destroyed by acting on the incorporated exo-sample nucleotide.Two embodiments are presented. In a first embodiment, the exo-sample nucleotide is incorporated by carrying out the amplification to reaction in the presence of an excess of exo-sample nucleotide tri-phosphate.In a second embodiment, the exo-sample nucleotide is incorporated by carrying out the amplification reaction in the presence of an oligonucleotide which has, as part of its sequence, one or more exo-sample nucleotides.

The invention belongs to the field of plant biotechnology, and particularly relates to a flanking sequence of a foreign insert segment in the maize genetic modification event IE034 and application of the flanking sequence. Testing by the Southern blot method show that an insertion side is present in the maize genetic modification event IE034, and a 5'-end flanking sequence of the foreign insert segment on the insertion site is showed as SEQ ID No.1. The invention provides a primer for detecting the flanking sequence, such as nucleotide sequences showed in SEQ ID No.4 and SEQ ID No.5. Testing of the flanking sequence of the foreign insert segment in the maize genetic modification event IE034 is applicable to detection of genetic modification IE034 of insect-resistant maize (including parents, hybrids F1 and offspring) and products of the insect-resistant maize ( including plants, tissues and seeds and products of these).

Methods for determining the presence of double stranded nucleic acids in a sample are provided. In the subject methods, nucleic acids present in a fluid sample are translocated through a nanopore, e.g. by application of an electric field to the fluid sample. The current amplitude through the nanopore is monitored during the translocation process and changes in the amplitude are related to the passage of single- or double-stranded molecules through the nanopore. The subject methods find use in a variety of applications in which the detection of the presence of double-stranded nucleic acids in a sample is desired, e.g. in hybridization assays, such as Northern blot assays, Southern blot assays, array based hybridization assays, etc.

Transgenic chloroplast technology could provide a viable solution to the production of Insulin-like Growth Factor I (IGF-I), Human Serum Albumin (HSA), or interferons (IFN) because of hyper-expression capabilities, ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification processing). Tobacco is an ideal choice because of its large biomass, ease of scale-up (million seeds per plant), genetic manipulation and impending need to explore alternate uses for this hazardous crop. Therefore, all three human proteins will be expressed as follows: a) Develop recombinant DNA vectors for enhanced expression via tobacco chloroplast genomes b) generate transgenic plants c) characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) large scale purification of therapeutic proteins from transgenic tobacco and comparison of current purification / processing methods in E. coli or yeast e) Characterization and comparison of therapeutic proteins (yield, purity, functionality) produced in yeast or E. coli with transgenic tobacco f) animal testing and pre-clinical trials for effectiveness of the therapeutic proteins. Mass production of affordable vaccines can be achieved by genetically engineering plants to produce recombinant proteins that are candidate vaccine antigens. The B subunits of Enteroxigenic E. coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are examples of such antigens. When the native LTB gene was expressed via the tobacco nuclear genome, LTB accumulated at levels less than 0.01% of the total soluble leaf protein. Production of effective levels of LTB in plants, required extensive codon modification. Amplification of an unmodified CTB coding sequence in chloroplasts, up to 10,000 copies per cell, resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as oligomers (about 410 fold higher expression levels than that of the unmodified LTB gene). PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that chloroplast synthesized CTB assembled into oligomers and was antigenically identical to purified native CTB. Also, GM1-ganglioside binding assays confirmed that chloroplast synthesized CTB binds to the intestinal membrane receptor of cholera toxin, indicating correct folding and disulfide bond formation within the chloroplast. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed. The introduced gene was stably inherited in the subsequent generation as confirmed by PCR and Southern blot analyses. Incrased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant based oral vaccines and fusion proteins with CTB needing oral administration a much more practical approach.

The invention provides a carrier incapable of generating frameshift mutation after recombination as well as a method and an application for gene fixe-point knock-in in a Xenopus laevis genome. The method comprises steps as follows: (1), guide RNA (ribonucleic acid), Cas9 nuclease and a donor carrier with a pancreas ela-fluorescent screening label and a Cas9 target fragment are contained in a fertilized egg of Xenopus laevis; (2) under the joint action of guide RNA and Cas9 nuclease, a target gene in the Xenopus laevis genome and the double-chain Cas9 target fragment on the donor carrier are shorn; (3), gene fixed-point knock-in of the Xenopus laevis genome is realized through a DNA (deoxyribonucleic acid) recovery function of Xenopus laevis cells; (4), G0-generation embryos are screened through the pancreas ela-fluorescent screening label, and F1 is subjected to southern blot identification. The carrier incapable of generating frameshift mutation after recombination as well as the method and the application for gene fixe-point knock-in in the Xenopus laevis genome lay a foundation for research of genetics and human diseases with Xenopus laevis as a model animal.

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

This invention provides a method for the development of novel plant germplasm using segregates from a transgenic line combined with PCR-based zygosity testing and, optionally, Southern blot analysis.

The present invention is directed to a method of Acacia mangium regeneration through organogenesis and a method of genetic transformation of Acacia mangium. The method of regeneration comprises inducing callus from different parts of seedlings and vegetatively micropropagated plantlets as explants; adventitious bud induction followed by pinnate leaf and bud elongation and eventually, elongated shoots were induced to root. Based on the regeneration system, a marker gene GUS under cauliflower mosaic virus promoter was introduced to Acacia mangium via Agrobacterium infection. GUS staining in the regenerated plants and Southern blot hybridization prove the incorporation of the foreign gene into the host genome and expression of the foreign gene.

The invention discloses a method for identifying the age of ginseng by utilizing the length of a ginseng telomere. The method comprises the following steps: (A) establishing a mathematical model: (a1) selecting a plurality of samples from the positions 1-2 cm below the rhizomes of the ginseng of known age and extracting genomic DNA from each sample; (a2) taking each genomic DNA as a template, conducting qPCR on a primer pair distinguished from the ginseng telomere to obtain a Ct value and marking the Ct value as T, and conducting qPCR on a primer pair distinguished from a single copy gene of the ginseng to obtain another Ct value and marking the Ct value as S; (a3) calculating the T / S ratio for each ginseng; (a4) taking the ages of the ginseng as the abscissa values and taking all the calculated T / S ratios as the ordinate values to draw a standard linear curve and obtain a curve equation; (B) adopting the mathematical model to identify the age of ginseng to be measured: obtaining the T / S ratio for the ginseng to be measured according to the operations from step (a1) to step (a3), and substituting the T / S ratio into the mathematical model to obtain the age of the ginseng to be measured. The method has the advantages that the identified age of ginseng is highly identical with the actual age of the ginseng, the cost is low, and the flux is high. Compared with the traditional Southern blot technology adopted for measuring the length of a ginseng telomere, the method provided by the invention has less demand for DNA and a shorter test period.

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a semi-dominant, single-locus mutation. It induces the accumulation of high levels of beta-carotene in various tissues that are normally devoid of carotenoids, turning them orange. Using a map-based cloning strategy, we identified a single gene representing Or and successfully verified its identity by functional complementation in the wild type cauliflower. The Or gene encodes a plastid membrane protein containing the DnaJ zinc figure domain. A likely gain-of-function mutation from a 4.3-kb retrotransposon insertion in the Or allele confers the orange phenotype in the mutant. Southern blot analysis revealed that Or is a single-copy sequence in the cauliflower genome. High level of expression of the Or gene and the protein was found in very young leaves, curds, and flowers at comparable abundance between wild type and the Or mutant. Or likely functions in regulating the differentiation of some non-photosynthetic plastids into chromoplasts, which provide the deposition “sink” for carotenoid accumulation. Successful demonstration of Or in conferring carotenoid accumulation in potato tubers indicates its potential use to improve the nutritional value in staple crops.