遗传学实验技术

E. coli K-12, being one of the best understood and thoroughly analyzed organisms, is the workhorse of genetic, biochemical, and systems biology research, as well as the platform of choice for numerous biotechnological applications. Genome minimization/remodeling is now a feasible app ...

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared p ...

Gene disruption methods have proved to be a valuable tool for studying gene function in yeast. Gene replacement with a drug-resistant cassette renders the disruption strain selectable and is stable against reversion. Polymerase chain reaction-generated deletion cassettes are de ...

We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then ...

The increasing genome sequence data of microorganisms has provided the basis for comprehensive understanding of organisms at the molecular level. Besides sequence data, a large number of experimental and computational resources are required for genome-scale analyses. Escher ...

Here we describe the systematic construction of well-defined, in-frame, single-gene deletions of all nonessential genes in Escherichia coli K-12. The principal strategy is based on the method for one-step inactivation of chromosomal genes in E. coli K-12 established by Datsenko and Wann ...

Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and puta ...

Genomic sequencing has transformed modern biology into an age of global analysis of gene expression, protein pathways, and metabolic networks. To understand wholecell function, biologists and bioinformaticians in many fields have developed a diverse set of methods and tools to iden ...

Genetic information consists of protein- and RNA-coding genes that exist in a range of sizes and noncoding cis- and trans-acting sequence elements. The use of long chromosomal deletion mutations is a powerful method for identifying essential genetic information through experiment ...

Antisense RNA technology has been used effectively to downregulate gene expression in a variety of bacterial systems. Regulated antisense RNA strategy provides an important approach to identify and characterize essential genes critical to bacterial growth in vitro and in vivo. This ...

Site-specific DNA photoaffinity labeling is a useful technique for mapping interactions of proteins with DNA in complex systems such as the yeast RNA polymerase III (Pol III) transcription complex, which consists of at least 25 different proteins (1,2). This technique allows probing of pr ...

The many detailed, though static, structures of protein-nucleic acid complexes have revealed the underlying structural determinants of the binding process: a high surface complementarity and a precise orientation of interacting roups. However, another important question not ...

Surface plasmon resonance (SPR) measures refractive index changes (Δn) at or near a surface and relates these to changes in mass at the surface Fig 1). This relationship is given by the Clausius Mossotti form (Eq. 2) of the Debye equation (Eq. 1): (1) Fig. 1.Schema showing the principle of surface plasmon res ...

Interactions of proteins with nucleic acids play important roles in biological phenomenon. Almost every stage in the regulation of gene expression involves the interaction of proteins with specific nucleic acids sequences. The identification of the nucleic acid recognition seq ...

Many plant pathogenic fungi differentiate a series of highly specialized infection structures to invade and colonize host tissues. Especially at early stages of infection, the ratio of fungal to plant biomass is very low. To investigate cell-specific patterns of gene expression, it is ne ...

Generation and characterization of mutants are important for the investigation of gene function. Gain-of-function technology is one of the most useful approaches for the systematic production of mutant resources. Full-length cDNAs have been collected from various plant species ...

Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the develop ...

Directed evolution is an often used approach toward new proteins with tailor-made properties. It consists of random variation of the coding sequence of a protein followed by an appropriate selection procedure or a suitable type of property read out. In many, if not all cases, it is of significant ad ...

The aim of this method is to guide a bench scientist to maximise cDNA library analyses to predict biologically relevant genes to pursue in the laboratory. Many groups have successfully utilised cDNA libraries to discover novel and/or differentially expressed genes in pathologies of inte ...

A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA norma ...