生物化学技术

Messenger RNA (mRNA) comprises approximately 1–5% of total cellular RNA. Although the actual amount depends on the type of cell and its physiological state, at any one time approximately 12,000 genes are being transcribed with approximately 500,000 mRNA molecules present in each mammali ...

A full analysis of the post-translational modifications that a given protein undergoes during transit through the secretory pathway may, in some cases, only be performed by analysis of the natural protein, expressed in its normal tissue.

The wheat germ extract in vitro translation system has been used widely for faithful and efficient translation of viral and eukaryotic messenger RNAs in a heterologous cell-free system (1–9). With respect to the yield of translation products, the wheat germ extract is less efficient than most ...

The identification of specific messenger RNA molecules and the characterization of the proteins encoded by them has been greatly assisted by the development of in vitro translation systems. These cell-free extracts comprise the cellular components necessary for protein synthes ...

Synthesis of specific RNA sequences in vitro is simplified because of the availability of bacteriophage RNA polymerases and specially designed DNA vectors. RNA polymerases encoded by SP6, T7, or T3 bacteriophage genomes recognize particular phage promoter sequences of their resp ...

In the last few years, a variety of polymerase chain reaction (PCR)-based mutagenesis procedures have been developed (1–16). Among these, the three-primer two-PCR methods (1–3) represented by the original “megaprimer” technique (1), appear to be the simplest and most versatile ones availa ...

Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been developed in order to achieve efficient mutagenesis of a target DNA sequence (1–8). This chapter describes an efficient and econ ...

In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships, gene expression and vector modification. Several methods for performing this technique have appeared in the literature (1–5). These procedures general ...

The ability to introduce specific changes into almost any given DNA sequence has revolutionized the analysis of cloned genes. This technique has enabled researchers to identify regions necessary for the regulation of gene expression. Also, it was and still is instrumental to learn about t ...

Site-directed mutagenesis provides a powerful means for probing protein structure and function. Using this approach, one can introduce specific amino acid changes at any given position in the protein sequence and test the functional consequences of these mutations in vitro or in vivo. If an ...

DNA undergoing electrophoresis in agarose assumes a conformation that only permits the movement of molecules up to about 20 kb in size. Beyond this limit, mobility rapidly decreases as the molecules become trapped in the agarose matrix. A reduction in agarose concentration to 0.5% and the appl ...

Exonuclease III (Exo III) will digest double-stranded DNA in a 3′ to 5′ direction if the DNA is blunt ended or possesses a 5′ overhang. It will not digest if there is a 3′ overhang of three or more bases, or if the 3′ end has had thiophosphate-containing bases incorporated into it. In order to generate a set of insert delet ...

Gene expression is controlled by cis-regulatory elements. Generally, the most important elements that are required for transcription are contained in the promoter sequences located upstream of a gene (1). Eukaryotic RNA polymerase requires several accessory factors, such as TFII ...

RNA in biological systems is associated with proteins. Recent work in eukaryotes has identified common motifs present in families of RNA-binding proteins. Usually, RNA-binding proteins recognize both sequence and structure at their target sites. Therefore, identification of pr ...

Determination of cellular phenotypes results from the expression of a limited number of genes whose products interact to establish a unique environment. The mechanisms by which individual cells can selectively express only a few of all the genes in a specific cell have been the focus of intense ...

The detection of specific nucleic acid species following electrophoretic separation of a complex sample may be undertaken by the use of Southern blotting (1). This technique immobilizes the separated DNA following its digestion with restriction enzymes. The separation is usually c ...

The two-hybrid system was originally devised by Fields and Song as a protein interaction detection system in yeast (1). Subsequently, it has been employed in many laboratories as a means of screening cDNA and genomic fusion libraries for protein interaction partners (2–8). The method relies on ...

The gel shift assay is one of the most powerful methods for the analysis of DNA-protein interactions (1,2). The assay itself is simple. DNA and protein are mixed together, the solution subjected to electrophoresis through polyaerylamide, and the gel is then analyzed for DNA, usually by autoradio ...

Dimethyl sulfate (DMS) is an effective and widely used probe for sequence-specific protein-DNA interactions. It is the only probe routinely used both for in vitro (methylation protection, methylation interference) and in vivo (DMS genomic footprinting) applications since it rapi ...

Agarose gel electrophoresis is generally adequate for resolving nucleic acid fragments in the size range of 100 nucleotides to around 10–15 kb (see Chapter 13). Below this range, fragments are both difficult to separate and hard to visualize because of diffusion within the gel matrix. These pr ...