遗传学实验技术

Whole mount in situ hybridization is a process that allows the visualization of gene expression (mRNA) within the cells of an intact organism. By comparing gene expression domains between organisms that have been subjected to different environmental conditions, an understanding of t ...

Differential display polymerase chain reaction (PCR) can facilitate the identification of novel molecular end points related to contaminant exposure in a wide range of species. To date, various differential display methodologies have been described in detail. Herein, we describe a ...

Nylon membrane-based cDNA macroarrays are a widely available alternative to cDNA microarrays for the collection of large-scale gene expression data. cDNA macroarrays are used in many areas of molecular biology research for applications ranging from gene discovery to gene express ...

Many organisms provide excellent models for studying disease states or for exploring the molecular adaptations that allow cells and organisms to cope with or survive different stresses. The construction of a cDNA library and subsequent screening for genes of interest allows researc ...

The method we describe in this chapter describes the synthesis and use of cDNA macroarrays for determining changes in gene expression due to environmental toxicants as well as the methods and materials that are required to do this work. While the details are for investigators working with nont ...

The use of DNA microarrays has gained wider acceptance as a standard tool for molecular biology studies over the past decade. In particular, biomedical studies embraced this technology as soon as arrays were produced for the common laboratory species. Slower to develop, however, has been the u ...

Environmental pollutants may affect the activities of many cellular enzymes. The effect on the proteome of enzymatic inhibitors can be determined using two-dimensional (2D) gel electrophoresis. In neuroendocrine cells, proprotein convertases 1 and 2 (PC1 and PC2) mediate the prote ...

Pollution in aquatic environment is of increasing concern for its impact on both human and natural populations. Applying proteomics to monitor marine pollution is a new approach to evaluate the effects of environmental pollutants on the biota. Aquatic organisms living in coastal and est ...

DNA methylation patterns are often altered in human cancer and aberrant methylation is considered a hallmark of malignant transformation. Several methods have been developed for the characterization of gene-specific and genome-wide DNA methylation patterns. In this chapter, we ...

A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA methylation in CpG islands. The basic concept relies on direct immunodetection of 5′ methylcytosines (5′ mCs) without the need for bisulfite treatment utilizing a microarray format. This sy ...

noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genom ...

The ability of sodium bisulfite to modify cytosines in a methylation-dependent manner allows the conservation of DNA methylation information during PCR amplification. PCR products amplified from bisulfite-modified DNA have significantly different base compositions ac ...

Fluorescent automated DNA sequencing based on ″one-lane, four-dye″ technology has played a pivotal role in the success of the human genome project. One Applied Biosystems 377 sequencer can produce at least two runs per day of sequence reads averaging 750 bases in length, each run produces up to 96 re ...

Three fundamental technologies have emerged in genetic analysis that have widespread and immediate benefits. DNA synthesis, fluorescence-based DNA analysis, and the polymerase chain reaction (PCR) are being integrated for a range of applications, from forensics to the understa ...

The development of polymerase chain reaction (PCR) (1), fluorescent DNA labels, and automated detection systems have revolutionized the efficiency, safety, and speed of DNA sequencing as compared to the original method of Sanger et al. (2), who published their chain terminating inhibit ...

DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from degradation by an enzyme or chemical reagent. Exonuclease III is a suitable probe to analyze the boundaries of a protein when it is necessary to el ...

Assaying sequence-specific DNA–protein complex formation in vitro often involves the use of specific labelling or modification of the components of the complex to provide unique signals that can be used to assess the affinity of the interaction. Surface plasmon resonance (SPR) spectr ...

Understanding the forces driving formation of protein/DNA complexes requires measurement of the Gibbs energy of association, ΔG, and its component enthalpic, ΔH, and entropic, ΔS, contributions. Isothermal titration calorimetry provides the enthalpy (heat) of the binding reac ...

Circular dichroism (CD) is a well-established technique for the analysis of both protein and DNA structure. The analysis of protein–nucleic acid complexes presents greater challenges, but at wavelengths above 250 nm, the circular dichroism signal from the DNA predominates. Examples ...

Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein–DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein–DNA complex and for free protein can be informative about the environment of ...