遗传学实验技术

A very powerful method of probing DNA-protein interactions is the technique of site-directed mutagenesis. Using this approach one can introduce specific amino acid changes at any given position in the amino acid sequence of a DNA-binding protein, and test the functional consequences of t ...

The basic principle of the DNA footprinting technique is the measuring of the accessibility of the DNA by a probe. The probe can be an enzyme or a chemical reagent that is able to cut the DNA backbone. The target is a DNA fragment with a signal-sequence for a sequence-specific binding protein. The sites on the DNA ...

Cassette mutagenesis is a technique for altering a protein sequence at the DNA level by replacing a section of genetic information with an alternative sequence, normally provided by a synthetic DNA duplex. First, the gene contained in a suitable vector is cleaved with two restriction enzymes. ...

The ability to express recombinant proteins at high level in bacteria has led to dramatic increases in our understanding of protein structure and function in recent years. These techniques have provided the means to isolate the substantial quantities of recombinant protein required f ...

Many DNA binding proteins are known to consist of a number of domains-discrete compact regions of a protein that often have distinct functional properties. Structural domains within a protein are generally indicated by limited proteolysis (see Chapter 13) and the existence of function ...

Chemical modification is a powerful tool for investigating the accessibility and function of specific amino acids within folded proteins. It has provided significant information regarding the role of different amino acids at the binding sites of numerous enzymes and DNA binding pro ...

It has long been known that the uranyl(V1) ion (UO2 2+) forms strong complexes with various inorganic and organic anions, including phosphates, and that the photochemically excited state of this ion is a very strong oxidant (1). For instance, uranyl-mediated photooxidation of alcohols has been ...

Structural studies of DNA-protein complexes have now made it clear that specific sequence recognition in these systems is accomplished in two ways, either directly by the formation of hydrogen bonds to base-pair edges from amino acid side chains located on a DNA-binding motif, such as a helix-t ...

The DNA double helix is highly malleable and when constrained, either as a small circle or by the action of a protein, can be readily distorted from its energetically favored conformation. Such distortions may be relatively moderate, as exemplified by smooth bending that maintains base stack ...

Within the last few years footprinting techniques have become increasingly important in the study of protein-nucleic acid interactions. This is partly the result of a fast-growing number of known nucleic acid binding proteins but also because of an increase in the available probes that can ...

Knockdown of cellular RNA using short interfering RNA has enabled researchers to perform loss-of-function (LOF) experiments in a wide variety of cell types and model systems. RNA interference techniques and reagents have made possible experiments that test everything from the analy ...

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Curre ...

DNA methylation is an epigenetic modification that plays a crucial role in the control of gene expression and chromosome structure in plants and mammalian cells. Multiple types of DNA fingerprinting techniques have been developed and applied to investigate DNA methylation profiles ...

Differential methylation hybridization (DMH) is a high-throughput DNA methylation screening tool that utilizes methylation-sensitive restriction enzymes to profile methylated fragments by hybridizing them to a CpG island microarray. This array contains probes spanni ...

Genomic representations using ligation-mediated PCR have been used successfully as the foundation for a number of high-throughput assays. HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) is an example of the use of such representations to study cytosine methylation ...

DNA methylation levels are affected by numerous environmental influences, including diet and xenobiotic exposure, and neoplasia has been firmly associated with genomic hypomethylation and localized hypermethylation of tumor suppressor genes. To reverse methylation-i ...

Amplification of sodium bisulfite-treated DNA is widely used to study DNA methylation. The proportion of methylated sequences of a specific DNA region in a sample can be determined by the analysis of PCR products or directly calculated from real-time PCR amplification of bisulfite-trea ...

Circulating extracellular nucleic acids derived from body fluids such as blood are commonly analyzed to assess malignant diseases. Efficient isolation, extraction, quantification, modification, and analysis methods remain important for utilizing circulating nucleic ...

The HeavyMethyl (HM) assay is a real-time PCR assay suitable for the qualitative and quantitative DNA methylation analysis of fresh, frozen, or formalin-fixed paraffin-embedded tissues and remote samples, such as serum, plasma, and urine. The HM uses a methylation-specific oligonucl ...

MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation in genomic DNA. MethyLight relies on methylation-specific priming combined with methylation-specific fluoresc ...