DNA recombinant

Two approaches to producing recombinant DNA for cloning have been developed for use with somewhat different applications  

Cloning DNA restriction fragments is usefijl in the following applications  

The first step in this procedure is to produce restriction fragments of DNA using restriction endonucleases. 

These enzymes are isolated from bacteria, their natural source. There are many different restriction endonucleases isolated from a variety of bacteria that are now readily available commercially. In bacteria they act as part of a restriction/modification system that protects the bacteria from infection by DNA viruses. 

This Is the DNA sequence recognized by the restriction endonuclease EcoRI. Rgure 1-6-1. EcoRI Recognition Sequence 

When scientists became concerned about whether this new technology posed risks to humans and the environment, there was an unprecedented (and 

The industrial use of rDNA technology includes the production of bleach-resistant enzymes that are used in laundry detergents to degrade 

Snpreme Conrt stated that a genetically engineered bacterium, designed to digest oil in oil spills by researchers at Exxon, could be patented. 

Hyone-Myong (1996). Enzymology Primer for Recombinant DNA Technology. San Diego Academic Press. 

Garrett, Reginald H., and Grisham, Charles M. (2002). Principles of Biochemistry With a Human Focus. Fort Worth, TX Harcourt College Publishers. 

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Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

Fig. 3. Constmetion of a recombinant DNA by joining vector and passenger fragments where (I I I I I I) represent sticky ends. Both A and B genes represent selectable traits, so that introduction of foreign DNA in B gene leads to the loss of an identifiable function. REP represents repHcation and...

In many cases it is possible to synthesize the product of a gene in a different organism, eg, bacteria, yeast, or higher eukaryote. Recombinant DNAs directing the synthesis of the gene product must contain information specifying a number of biochemical processes. 

Replication of the Recombinant DNA. In bacteria, repHcation of the recombinant DNA is provided by origin sequences, derived usually... 

Methionyl hGH. The first form of hGH to be produced through recombinant DNA technology was actually a derivative of hGH having one additional methionine residue at its N-terminus (11). Although technology has advanced to the stage where natural sequence hGH can easily be produced, as of this writing this derivative, referred to as methionyl hGH, is stiU produced commercially. 

Point Mutations. Since the advent of recombinant DNA technology, a number of researchers have used point mutation techniques either to delete one or more residues within the hGH molecule or systematically to change from one amino acid to another to probe hGH stmcture/function relationships (33). 

Human growth hormone was originally manufactured by isolation of the natural product from human pituitaries and subsequent purification of the protein. Since 1985, manufacture of hGH has been almost exclusively by recombinant DNA technology. 

Human growth hormone is one of the largest selling therapeutic proteins produced by recombinant DNA technology. Annual worldwide sales increased from 130,000,000 in 1987 to 575,000,000 in 1992 (47). Upon approval of additional indications, the sales of hGH are expected to increase even more. 

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. 

Biotechnology is being appHed in the dairy industry. A significant and controversial development is the technique of producing transgenic animals, ie, animals in which hereditary deoxyribonucleic acid (DNA) has been augmented by DNA from another source, using recombinant DNA (rDNA) techniques. 

A fermentation route to 1-butanol based on carbon monoxide employing the anaerobic bacterium, Butyribacterium methjlotrophicum has been reported (14,15). In contrast to other commercial catalytic processes for converting synthesis gas to alcohols, the new process is insensitive to sulfur contaminants. Current productivities to butanol are 1 g/L, about 10% of that required for commercial viabiUty. Researchers hope to learn enough about the bacteria s control mechanisms to be able to use recombinant DNA to make the cells produce more butanol. 

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