生物化学技术

Ketomethylene peptide isosteres have been the focus of a variety of synthetic studies because of their potential as therapeutic agents in the treatment of medical conditions mediated by proteases (1–3). The dipeptide core units 1 are chemically characterized by a 1,4-disposition of the c ...

Peptidomimetics have become immensely important for both organic and medicinal chemists (1). The alteration of peptides to peptidomimetics has included peptide side chain manipulations, amino acid extensions (2), deletions (3), substitutions (1a,b), and most recently backbone ...

With the discovery of an increasing number of biologically active peptides and peptide mimetics (1–3), there is a pressing need for the development of strategies to deliver these biologically active compounds to the desired site of action. The preceding two chapters have described two meth ...

One of the major obstacles to the development of biologically active peptides as clinically useful therapeutic agents has been their low permeation through biological barriers (e.g., intestinal mucosa, blood-brain barrier) and their metabolic lability (1,2). Overcoming these pr ...

The purpose of this technique is the detection and characterization of specific DNA sequences. The DNA is fragmented by digestion with a restriction endonuclease, and the fragments separated by agarose gel electrophoresis. The DNA is then denatured in the gel and transferred to a nitrocel ...

Autoradiography of gels containing a 32P label is used to detect and quantitate the radioactive label in a particular band or spot. Since 32P is a high energy β-emitting isotope, no fluor is required in the gel (as in fluorography) to increase the efficiency of detection. However, since most of the radia ...

This procedure yields thin flakes of freeze-dried material from tissue culture cells. Up to 0.5 g of wet cells can be processed at one time. Dried cells, stored indefinitely at −20�C, have full lactate dehydrogenase activity (1) and DNA polymerase activity (2). The dried cells stored for a few days at room t ...

The isolation of high molecular weight eukaryotic DNA in good yield is an important prerequisite for the analysis of specific sequences by Southern blotting (Chapter 9), or for molecular cloning in phage or cosmid vectors (Chapter 49).

Dideoxy chain termination DNA sequencing was developed by Sanger and colleagues (1, 2) and is a simple and extremely accurate method of obtaining thousands of bases of sequence data per day. The procedure requires a single-stranded DNA template and a primer complementary to the 3′ end of the region ...

As mentioned in Chapter 51, DNA sequence analysis is based on high-resolution electrophoresis on denaturing polyacrylamide gels. In both the partial chemical cleavage method (Chapter 51) and the partial resynthesis method (Chapter 53), the labeled DNA fragments to be separated have one ...

Unlike the determination of the amino acid sequence of proteins and peptides, which is based on the sequential degradation of these structures and the subsequent identification of the cleaved-off amino acid residue, DNA sequence analysis is based on the high-resolution electrophore ...

The manipulation of gene sequences between cells is a fundamental technique in genetics. Mammalian cells will take up and express genes when they are exposed to either metaphase chromosomes or naked genomic or recombinant DNA. In each case the uptake and expression is enhanced by the formation ...

The term satellite DNA is used for a DNA component that gives a sharp band in a density gradient and can be resolved from the broader main band of DNA in the gradient. The usual gradient material is CsCl in aqueous buffer and the Cs+ ions form a density gradient in a centrifugal field. DNA in the solution sediments to i ...

Bacteriophage lambda contains a double-stranded DNA molecule of about 50 kilobases (kb). This molecule is linear in the phage particle and it possesses a set of single-stranded complementary ends (the cos region) by which the molecule is joined to form a circular molecule in the first step of the in ...

Having introduced a recombinant DNA molecule to be analyzed into either a minicell or a maxicell, it is then possible to detect the gene products encoded for by this DNA by incubation of a minicell or maxicell preparation with radioactively labeled precursors of RNA and protein; uridine or uracil in ...

The maxicell system (1) is based on the following observation: The number of lesions induced with UV light in a DNA molecule is proportional with the size of the molecule and the dose of the UV light. Since most plasmids are less than 1/1000 the size of the chromosome and are usually present in 10–100 copies per ce ...

The term minicell was introduced by Adler et al. (1) for the small spherical cells produced by abnormal cell division at polar ends of certain Escherichia coli cells. The phenomenon itself however, had already been described as early as 1930 (2) for a strain of Vibrio cholera. These minicells do not cont ...

After ligation and transformation, a number of clones will (it is hoped) be obtained that can be identified as recombinants by the occurrence of insertional inactivation, by analysis of small-scale plasmid preparations, or more specifically by in situ hybridization. An additional stra ...

There are a number of methods available for screening either potential recombinant clones or natural isolates for the possession of plasmids. The method described in this chapter is based on that described by Sherratt (1) and is the simplest and most rapid of these. The name arises from the fact that t ...

Phage vectors are often used rather than plasmids, particularly for the production of gene “banks” or “libraries.” The plaques produced can be screened for the presence of specific DNA sequences by hybridization using a procedure similar to that used for colony hybridization (see Chapter 4 ...