生物化学技术

Highly selective and specific protein-protein and peptide–protein interactions are elicited by a large number of natural specific biological responses. The mimicry of these natural interactions has been an important and useful strategy for the development of metabolically st ...

The clinical development of orally active peptide drugs has been limited by their unfavorable physicochemical characteristics (e.g., charge, hydrogen bonding potential, size), which prevent them from permeating biological barriers such as the intestinal mucosa, and also their l ...

As will be evident from a number of the following chapters (i.e., Chapters 31, Chapters 38–Chapters 41, Chapters 51–Chapters 53), gel electrophoresis of DNA is a widely used technique in molecular biology. In a number of cases, e.g., for such procedures as cloning and DNA sequencing, it is not sufficient j ...

The procedure described in this chapter is used to display single-stranded nucleic acids according to their sizes, within the range of 0.5 to 30 kilobases (kb). Possible applications include examining products of in vitro synthesis, hybrid-selected RNAs from total cellular nucleic aci ...

This article details two methods for separation and visualization of RNA under nondenaturing conditions, i.e., where the secondary structure of the molecules is left intact during electrophoresis. The first method describes electrophoresis in a 2% (w/v) agarose gel in a dilute, neutral p ...

This method of RNA extraction relies on a relatively gentle lysis procedure that should burst the cells, but leave the nuclei intact. Contamination of a relatively RNase-free cytoplasmic environment with nuclear nucleases is thus minimised. Next the polysomes are dissociated with SDS a ...

Successful extraction of RNA depends on the quantitative recovery of pure nucleic acids in an undegraded form. In practice, this means that a selective extraction process is required to remove all the unwanted cellular material in a manner that minimizes degradation of the RNA by hydrolysis or ...

The vast majority of eukaryotic mRNA molecules contain tracts of poly(adenylic) acid, up to 250 bases in length, at the 3′ end. This property is very useful from the point of view of mRNA extraction because it forms the basis of a convenient and simple affinity chromatography procedure (1). Under high salt ...

This method relies on the strong chaotropic nature of the reagents involved to completely denature any ribonuclease (RNase) present in the sample. After lysis in guanidine thiocyanate buffers there are two possibilities for isolation of the RNA. One method involves a series of different ...

A DNA-directed cell-free protein synthesizing system was originally developed by Zubay (1). The system contains a crude extract prepared from Escherichia coli. This extract contains the machinery necessary for the transcription and translation, i.e., ribosomes and RNA polymerase. ...

The sequence of poly(adenylic) acid, present at the 3′ end of the majority of eukaryotic mRNA molecules, forms the basis of a sensitive technique for the estimation of mRNA content in nucleic acid samples. Under suitable conditions, poly(A) will form RNA-RNA hybrids with poly(U) in vitro. The poly (A) co ...

The separation of RNA on the basis of size by sucrose gradient fractionation is a technique frequently employed in a cloning strategy (1–3). After production of poly(A)+ mRNA by affinity chromatography (see Chapter 16) the RNA can be fractionated once or twice on sucrose gradients to produce a subp ...

The Burton assay for DNA is a colorimetric procedure for measuring the deoxyribose moiety of DNA. It is reasonably specific for deoxyribose, although very high concentrations of ribose (from RNA) or sucrose must be avoided. The method can be used on relatively crude extracts and in other circums ...

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 synthe ...

The entire complement of in vitro translation products derived from a mRNA population may be analyzed by polyacrylamide gel electrophoresis followed by fluorography and autoradiography. It is often necessary however to demonstrate the synthesis of a polypeptide translation pr ...

The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method (1–3). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provid ...

Isolated nuclei will continue synthesis of RNA initiated in vivo, but reinitiation of synthesis is rare in washed nuclei (1). This situation can be exploited to measure instantaneous rates of in vivo transcription because the cell-free conditions are well-defined and nascent transcri ...

For the initial characterization of a recombinant plasmid, it is necessary to determine the size of the plasmid or, preferably, the size and characteristics of the insert itself. A method is therefore required for the simultaneous preparation, from a number of isolates, of plasmid DNA in a state su ...

The cleared lysate method (see Chapter 25) is not usually very effective for isolation of plasmids larger than about 20 kb. Recovery of plasmid DNA is often poor, presumably because high molecular weight plasmids are removed by the clearing spin. An alternative procedure, therefore, is to load the ...