Aida T.,Tokyo Medical and Dental University |
Chiyo K.,Tokyo Medical and Dental University |
Usami T.,Laboratory of Recombinant Animals |
Ishikubo H.,Tokyo Medical and Dental University |
And 8 more authors.
Genome Biology | Year: 2015
Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools. © 2015 Aida et al.; licensee BioMed Central. Source
Fujita Y.,Japan National Agriculture and Food Research Organization |
Murakami K.,Kagawa Prefectural Agricultural Experiment Station |
Haraguchi H.,Fasmac Co.
Nippon Shokuhin Kagaku Kogaku Kaishi | Year: 2012
We compared the performance of three DNA extraction methods using 12 processed wheat foods, e.g. noodles, bread, and confectionery items. Three commercial kits, DNeasy Plant Mini Kit, Genomic-tip 20/G, and GM quicker 3, were each tested in three laboratories. Of the three methods, Genomic-tip 20/G produced the best results for DNA purity testing when applied to castella, and it was generally well-suited to all of the food types tested. However, all of the methods successfully extracted DNA from all the analyzed foods, and the DNA was suitable for PCR amplification using a wheat-specific marker. However, PCR amplification was not reproducible in some of the processed food samples when we applied a DNA marker for cultivar identification, regardless of the method used. The results show that, depending on the type of food being tested, PCR conditions need to be optimized slightly in ordertoidentifyacultivar accurately. Source
Takabatake R.,Japan National Agriculture and Food Research Organization |
Akiyama H.,Japan National Institute of Health Sciences |
Sakata K.,Japan National Institute of Health Sciences |
Onishi M.,Fasmac Co. |
And 7 more authors.
Journal of the Food Hygienic Society of Japan | Year: 2011
A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (Cf), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined Cf values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD R), and the determined bias and (RSDR) values for themethod were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12. Source
Masamura N.,House Foods Group Inc. |
Kikuchi R.,Fasmac Co. |
Nagatomi Y.,Fasmac Co.
Bunseki Kagaku | Year: 2014
We developed a method to identify plant materials by amplifying by PCR the internal transcribed spacer 1 (ITS 1) region located between the 18S ribosomal RNA (18S rRNA) and the 5.8S ribosomal RNA (5.8S rRNA) genes using plant universal primers designed on the two genes, and determining the nucleotide sequence of the amplified product. The primer designed on the 18S rRNA gene is universal to a wide range of plant species, designed from a nucleotide sequence not found in fungi or yeast, and thus is applicable to plant materials contaminated with fungi/yeast. The current method correctly identified 36 of 45 commercially available plant species, including 9 plants identified up to the genus level. Onion scale leaves heated at 105 °C for 16 hrs and onion scale leaves contained in retort sauce were also identified. The method was then tested for its ability to identify possible contaminants in food, including peanut seed coat, calyxes of apples and pumpkins, bamboo, wood pieces and the pericarp of frozen eggplants. PCR products were obtained from the peanut, pumpkin and apple materials, and these plants were identified up to the species or genus level. These results suggest the potential of the current method for identifying plant contaminants in food. © 2014 The Japan Society for Analytical Chemistry. Source
Oguchi T.,Japan National Agriculture and Food Research Organization |
Oguchi T.,University of Tsukuba |
Onishi M.,Fasmac Co. |
Mano J.,Japan National Agriculture and Food Research Organization |
And 5 more authors.
Journal of the Food Hygienic Society of Japan | Year: 2010
A novel multiplex PCR method was developed for simultaneous event-specific detection of four events of GM maize, i.e., DAS-59122-7, MIR604, MON88017, and MON863. The single laboratory examination of analytical performance using simulated DNA mixtures containing GM DNA at various concentrations in non-GM DNA suggested that the limits of detection (LOD) of the multiplex PCR method were 0.16% for MON863. MIR604, and MON88017, and 0.078% for DAS-59122-7. We previously developed a nonaplex (9plex) PCR method for eight events of GM maize, ie., Bt11, Bt176, GA21, MON810, MON863, NK603, T25, and TC1507. Together with the nonaplex PCR method, the newly developed method enabled the detection and identification of eleven GM maize events that are frequently included in commercial GM seed used in Japan. In addition, this combinational analysis may be useful for the identification of combined event products of GM maize. Source