Recombinant-DNA techniques

Recombinant DNA Techniques.—Introduction. Although activity in the area of recombinant DNA has probably increased rather than diminished over the past two years, much of the work has been in consolidation and exploitation of the techniques developed at such a frantic pace during the late 1970 s. These were dealt with, in an inevitably cursory fashion, in the last volume. Two excellent small introductory books on the subject were published in 1980, and one issue of Science was largely devoted to articles on recombinant DNA. In addition, the first two volumes of a series of multi-author monographs on the principles and methods of genetic engineering appeared in 1980.  

The safety (or biological containment) of these phage vectors have also been increased by introduction of amber mutations so that they require suppressor strains of E. coli as hosts. 

Derivatives of phage A are able to act as vectors for much larger segments of DNA than is possible with plasmid vectors. However, the recombinant DNA must be packaged into the phage for amplification. Methods of in vitro packaging have been developed to yield up to 10 plaques per ftg of recombinant DNA. The in vitro packaging technique has permitted the construction and exploitation of totally novel vectors termed cosmids. These are plasmids into which the A cos site, at which 

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NPH Isophane Human Insulin Suspension. NPH isophane insulin, also called Humulin N, Insulatard NPH Human, or Novolin N is an intermediate-acting form of human insulin produced by recombinant DNA techniques. Mixtures Humulin 70/30 and Novolin 70/30 contain 70% NPH isophane and 30% regular, whereas Humulin 50/50 contains 50% NPH isophane and 50% regular. It is adrninistered subcutaneously and should not be given intravenously. Absorption is delayed because the insulin is conjugated with protamine in a complex of reduced isoelectric solubiUty. Therapeutically, this preparation is probably comparable to purified porcine NPH insulin. However, human NPH insulin may have a slightly shorter duration of action than comparable purified porcine products. 

It is often important to control the CSD of pharmaceutical compounds, eg, in the synthesis of human insulin, which is made by recombinant DNA techniques (1). The most favored size distribution is one that is monodisperse, ie, all crystals are of the same size, so that the rate at which the crystals dissolve and are taken up by the body is known and reproducible. Such uniformity can be achieved by screening or otherwise separating the desired size from a broader distribution or by devising a crystallization process that will produce insulin in the desired form. The latter of these options is preferable, and considerable effort has been expended in that regard. 

Recombinant DNA techniques have provided tools for the rapid determination of DNA sequences and, by inference, the amino acid sequences of proteins from structural genes. The number of such sequences is now increasing almost exponentially, but by themselves these sequences tell little more about the biology of the system than a New York City telephone directory tells about the function and marvels of that city. 

The specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

Experimental approaches that have afforded major insights to the processes described in this chapter include (1) use of yeast mutants (2) application of recombinant DNA techniques (eg, mutating or eliminating particular sequences in proteins, or fusing new sequences onto them and (3) development of in vitro... 

R. L. Rodriguez and R. C. Tait, Recombinant DNA Techniques An Introduction, Addison-Wesley Publishing, Reading, MA, 1983. 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

In approaching the study of the molecular mechanisms of heredity, this chapter first discusses the structural and functional roles of the genetic material, DNA. This includes an analysis of its replication and susceptibility to mutation. The health-related aspects of the use of recombinant DNA techniques are considered, and examples of then-use in the analysis of several human genetic diseases are used to illustrate the biochemical side of genetics. 

The Genetic Engineering (GE) tools and techniques are used to modify the genetic material of microorganisms, in very specific manners, to introduce or improve their capabilities towards a given industrial interest. From recombinant DNA techniques, new DNAs sequences are derived by the changes on other DNA strands. 

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