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Free keywords:
Molecular Cloning; Functional Expression; Heterogeneous Mixture; Complex Genome; Bacterial Host
Abstract:
Biochemical, biophysical and genetic studies of DNA segments of complex genomes are greatly facilitated by a variety of techniques, called molecular cloning of DNA, which permit propagation of single DNA segments of virtually any origin in bacterial cells. Molecular cloning requires in vitro recombination of DNA fragments with a prokaryotic genetic element (a plasmid or a bacteriophage DNA) which serves as replication vehicle (also called vector) in the bacterial host. The experimental conditions allow the production of bacterial clones each harboring a single fragment of exogeneous DNA out of an initially highly heterogeneous mixture of fragments. The following steps are involved: foreign DNA is first dissected into small pieces of up to some 17 000 basepairs in length. The fragments are then joined in vitro to the vector molecules by means of well characterized DNA enzymes. The resulting recombinant molecules are introduced into the host cells by a transformation or transfection step. Among the progeny of transformed or transfected cells those clones are selected which carry a fragment of interest. Selection is in most cases accomplished by a combination of genetic and physical methods and is based on properties of the vectors as well as on attributes of the cloned foreign DNA. It is anticipated that bacterial host cells are not only suitable for amplifying DNA but also for the expression of useful functions which originate from other, preferably higher organisms. Two questions cannot be answered conclusively at present: first, is functional expression of genes generally possible in a heterologous cellular environment, and second, if it is possible, is it always harmless or does it create, at least occasionally, a biological hazard. Besides a detailed description of the techniques developed for molecular cloning, problems connected with functional expression and biohazards are discussed. In addition, results are presented which were obtained in the recent past by applying DNA cloning procedures.
