RESUMEN
Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.
Asunto(s)
Clonación Molecular/métodos , ADN/química , ADN/genética , Secuencias Invertidas Repetidas , Escherichia coli/genética , Genes de Plantas/genética , Vectores Genéticos/genética , Ligasas/metabolismo , Mutagénesis , Plásmidos/genética , Interferencia de ARN , Factores de TiempoRESUMEN
Band-edge liquid crystal lasers are of interest for a number of applications including laser projection displays. Herein, we demonstrate simultaneous red-green-blue lasing from a single liquid crystal sample by creating a two-dimensional laser array fabricated from dye-doped chiral nematic liquid crystals. By forming a pitch gradient across the cell, and optically pumping the sample using a lenslet array, a polychromatic laser array can be observed consisting simultaneously of red-green-blue colors. Specifically, the two-dimensional polychromatic array could be used to produce a laser-based display, with low speckle and wide color gamut, whereby no complex fabrication procedure is required to generate the individual 'pixels'.
Asunto(s)
Color , Presentación de Datos , Rayos Láser , Cristales Líquidos/química , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, approximately 15%-20% represent putative homologs with a conservative cutoff of p < 10(-9), thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets.