遗传学实验技术

In 1992 Gavrieli et al. published a seminal article showing that apoptotic cells could be detected by an in situ assay (1). The labeling method depends on the ability of terminal deoxyribonucleotidyl transferase to add nucleotides to breaks in DNA, and has generally been termed TUNEL (terminal d ...

Specific DNA breaks underlie many morphological changes in normal and damaged cells. They can serve as important markers in cell and tissue research. Yet historically, the labeling of DNA breaks in situ was most often limited to the identification of apoptotic cells. Consequently, the major ...

DNA fragmentation is often used as a marker of programmed cell death in tissue sections. It is a sensitive indicator of apoptosis, particularly when utilized in combination with morphological verification. At least two types of DNA breaks are present in apoptotic cells: 1. Breaks with 3′OH and 5′ ...

Over the past decade, the comet assay, or single cell gel electrophoresis (SCGE) has become one of the standard methods for assessing DNA damage, with applications in genototoxicity testing, human biomonitoring and molecular epidemiology, ecotoxicology, as well as fundamental rese ...

The comet assay is a gel electrophoresis method that is used to visualize and measure DNA strand breaks in individual cells using microscopy. In its simplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution to remove lipids and proteins, and exposed to a weak elect ...

The assessment of cellular DNA damage is crucial in many areas of biology including immunology, developmental biology, aging, cancer, and environmental science. A variety of experimental techniques including alkaline sucrose gradient centrifugation, alkaline elution, nuc ...

The basic repeating unit of chromatin, the nucleosome, is known to play a critical role in regulating the process of gene transcription. The positioning of nucleosomes on a promoter is a significant determinant in its responsiveness to gene-inducing signals. For example, positioning and s ...

Chromatin immunoprecipitation coupled to DNA microarray has become a widely used method to study transcription factors and chromatin structure. Here, we provide a detailed protocol for the localization of the variant histone Htz1 in the S. cerevisiae genome. This protocol can easily be a ...

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the inte ...

The ensemble of the genes in the mammalian genome is organized into a structure of DNA and proteins known as chromatin. The control of gene expression by the proteins that bind to chromatin regulates many cell processes, such as differentiation and proliferation. Transcription of protein-e ...

We describe methods for the genetic analysis of a DNA–protein interaction from any species. The DNA-binding domain of the protein of interest is expressed in yeast cells as a fusion with a known transcriptional activation domain, and the target binding site is used as an artificial upstream acti ...

DNA-transcription factor interactions in eukaryotic systems have been documented by a broad gamut of biochemical techniques including deoxyribonuclease I (DNase I) footprinting and Southwestern (SW) assays. In spite of their wide applicability, each of these approaches prov ...

Various methodologies have been developed for the detection of DNA-binding activities and the identification of the “footprints” of a protein on DNA. The most widely used footprinting techniques employ reagents such as deoxyribonuclease I (DNase I) and dimethyl sulfate (DMS) for prot ...

A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein–nucleic acid complexes ...

The nucleosome and other chromatin complexes are examples of complicated protein–DNA assemblies that are not easily studied by traditional structural methods. Site-directed cleavage of DNA is a method for mapping the location of interaction of a specific site in a protein such as a linker h ...

The in cellulo analysis of DNA protein interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to g ...

Despite their rather recent invention, atomic force microscopes are widely available commercially. AFM and its special modifications (tapping mode and noncontact operation in solution) have been successfully used for topographic studies of a large number of biological objects ...

This method aims at providing structural information on protein or nucleoprotein complexes by high-resolution electron microscopy. The objective is to promote the self-assembly of the macromolecules into two-dimensional crystals in order to use electron crystallography me ...

The synthesis of 8-azido-2′-deoxyadenosine-5′-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography and UV spectroscopy. Its photoreactivity upon UV irradiation was studied. After incorporation of this dATP analog by nick t ...

Static site-specific protein–DNA photocrosslinking permits identification of protein–DNA interactions within multiprotein–DNA complexes. Kinetic site-specific protein–DNA photocrosslinking – involving rapid-quench-flow mixing and pulsed-laser irradi ...