Recombinant plasmids, multiplied several n treated with restriction endonuclense used i
Recombinant plasmids, multiplied several n treated with restriction endonuclense used i iljion times, are i (2) and (3)
Fig. 4-2 Steps m obtaining recombinant DNA In parallel, DNA (genome DNA or cDNA from virion RNA or mRNA) fiom a virus (1) is cut Enlo fragments by a selected restriction endonuclease
(2), and the circular DNA molecule of the plasm id vector is cut with the same endonuc lease (3) The viral DNA is inserted and ligated into the plasmid DNA, which is thus circularized agai n (4) The plasmid is then introduced into the host bacterium by transformation Ci) Replication of the plasmid as an episome (6) may produce many copies per bacterial cell (for small plasmids), or there may be only one copy (for Luge plasmids) Bacteria containing the desired plasmid are identified, cloned, and allowed lo grow (7) The plasmids are isolated from the bacteria and the viral DNA insert is excised (8) using the same restriction endonuclease employed in steps (2) and
(3) In this way a specified gene may be replicated several millionfold With appropriate genetic engineering including the use of regulatory and termination sequences, the protein pioduct of the inseited gene may be expressed in prokaryotic or eukarvotic cells
5. Marker rescue by transfection with gene fragments, as a method of genetic mapping and of site-specific mutagenesis 6 Production of proteins coded by specific viral genes using bacterial, yeast, insect, and animal cell expression systems, or by cell-free translation 7. Synthesis of peptides based on DNA sequence data
Genetic Analysis of Viruses (hat Cannot be Cultured
Growth in cell culture is not essential for the study of viral nucleic acids and proteins, since the introduction oi gene cloning and then the polymerase chain reaction (PCR) has made it it possible to produce virtually unlimited quantities of any required nucleic acid Remarkable progress has been made in the genetic analysis of some noncullivable viruses, such as the papillomaviruses, by using DNA extracted directly from papillomas. A recent milestone in the history of virology was the molecular cloning followed by determination of the complete nucleotide sequence of the hepatitis C virus genome without the virus ever having been seen by electron microscopy, let alone cultured (see Chapter 26).
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