生物化学技术

The cleared lysate method of plasmid isolation is commonly used to extract relatively small plasmids (up to about 20 kb) from gram-negative bacteria such as Escherichia coll. It relies on a very gentle lysis of the bacteria to release small molecules, including very compact, supercoiled plas ...

This chapter describes a simple and rapid way of extracting and purifying chromosomal DNA from E. coli and many other species of bacteria. This procedure is essentially a simplified version of that described by Marmur in 1961 (1). The cells are lysed by treatment with a detergent and the mixture is depr ...

Lambda, a temperate bacteriophage of E. coli, has two alternative modes of replication in sensitive cells, known as the lytic and lysogenic cycles. In the lytic cycle, after the lambda DNA enters the cells, various phage functions are expressed that result in the production of a large number of mature ...

The isolation of phage DNA from a purified phage preparation simply involves the removal of the proteins of the phage particle. This is done most easily by phenol extraction (Method A). The large amounts of high quality DNA needed for use as vectors or for markers for gel electrophoresis can be obtained ...

The fluorescence assay of Kissane and Robins (1) is used to quantify deoxypurine nucleosides in crude mixtures. Acid-catalyzed depurination exposes the 1′ and 2′ carbons of deoxyribose, which can then form a strongly fluorescent compound with diaminobenzoic acid (DABA). DABA can react wi ...

The discovery of the mode of action of the class of bacterial enzymes known as restriction endonucleases provided the major breakthrough in opening up the field of genetic engineering. In vivo, these enzymes are involved in recognizing and cutting up foreign DNA entering the cell; their most li ...

Since they are involved in such important processes as DNA replication, DNA repair, and DNA recombination, DNA ligases can be found in all living cells. Two prokaryotic DNA ligases have become indispensible tools in the fields of in vitro DNA recombination and DNA synthesis. DNA ligase from E. coli is a ...

Linearized plasmid vector molecules produced by restriction enzyme digestion can be reformed into circles by the action of DNA ligase. This is the most favorable reaction even when foreign DNA fragments are present. Thus, unless a direct selection technique is available, the reformed par ...

The principle of this procedure is very similar to that of the standard calcium chloride method, (see Chapter 34) i.e., exponential phase cells are harvested and washed with a solution containing divalent cations. This renders the cells competent, which simply means they are now able to take up DNA f ...

In the normal growth cycle of bacteriophage lambda, the proteins that ultimately form the head of the phage particle are assembled into an empty precursor of the head (prehead); the phage DNA is replicated separately and then inserted into the empty head particles—a process known as packaging. A n ...

Labeling of DNA fragments at the 5′ end with polynucleotide kinase is one of the methods currently employed to obtain highly labeled DNA for sequencing purposes. This end-labeled DNA is used in the partial chemical degradation method for sequencing DNA (see Chapter 51 and ref. 1).

Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3′-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). DNA polymerase 1 from E. coli ...

Characterization of RNA molecules by electrophoresis or hybridization frequently requires nucleic acid concentrations over 1 mg/mL. High molecular weight DNA in a mixture of nucleic acids limits the solubility and interferes with electrophoresis. DNase treatment makes the mi ...

The efforts to localize genes to human chromosomes date back to the early 1970s. Although few techniques were available to map genes, many scientists recognized that the ability to determine the location of genes and DNA sequences on human chromosomes would not only facilitate the identific ...

Nonradioactively labeled probes offer several advantages compared to radioactive ones, as they show long stability, high morphological resolution, and rapid developing time. There are different types of nonradioactive labeling methods available, although digoxigenin- ...

The first step for a successful in situ hybridization is the fixation of the tissue. This will ensure target nucleic acid retention and preservation of the tissue morphology. Either crosslinking or precipitative fixatives can be used, and a preference for either of the two types of fixative has o ...

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in Escherichia coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is ind ...

Many systems have been developed for the heterologous expression of recombinant proteins. They are often based on the fusion of the protein of interest with a naturally occurring protein (glutathione S-transferase , maltose binding protein , or protein A ) and using their natural affinity to ...

The heterologous expression of recombinant proteins is a valuable tool in the study of gene expression, and has resulted in the development of many systems to express and purify hybrid proteins. Most of these systems are based on the fusion of the protein of interest with a naturally occurring prot ...

The prototype baculovirus is the Autographa californica nuclear polyhedrosis virus (AcNPV), which has been the focus for most studies developing expression vector systems. In contrast to other reviews on this topic (1–3) this chapter aims to describe the various practical aspects of the ...