DNA recombinant technology

cntting DNA at precise locations to yield the DNA segment (gene) of interest by use of sequence specific restriction endonuclease (restriction enzyme)  

selecting a proper vector and cutting the vector preferably with the identical restriction enzyme  

construction of the recombinant DNA by inserting the DNA segment of interest into the vector by joining the two DNA fragments with DNA ligase  

introduction of the recombinant DNA into a host cell that can provide the enzymatic machinery for DNA replication  

selecting and identifying those host cells that contain recombinant DNA  

Recombinant DNA Technology.—Composite DNA molecules formed of a vector and an introduced segment of foreign DNA are called chimeras. They can be formed in a number of ways  

The projecting 3 -single strand ends which are left serve as a primea for the enzyme terminal transferase which is used to put a homopolymer of one nucleotide on the vector DNA and a homopolymer of the complementary nucleotide in the foreign DNA. The two DNA segments can then be annealed together, any gaps are filled by the action of DNA polymerase and the chimera is sealed with DNA ligase. 

3 bases of the anticodon according to the AU-pairing and GC-pairing. However, the 3 base of the codon may interact with the 5 base of the anticodon with a certain pattern of redundancy known as the wobble hypothesis  

The anticodon can be found in the middle of the unpaired anticodon loop (7 nucleotides) of tRNA ( 76 nucleotides) between positions 30 and 40 (at around position 35). The anitcodon is bordered by an unpaired pyrimidine, U, on the 5 side and often an unpaired alkylated purine on the 3 side. 

A good cloning vehicle must generally have a number of features  

Considerable effort has been spent on developing the vectors (for example pBR322) so that they incorporate desirable features. These include  

Enhanced gene expression can be achieved by the addition of a promoter to the cloned gene. For example, the lac promoter may be added upstream of the protein DNA sequence, giving rise to the expression of the protein in the presence of lactose and in the absence of glucose, (cf. operon hypothesis Section S.7). 

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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. 

Recombinant DNA technology has already provided several products of therapeutic interest from mammalian cells. Table 2 gives examples of products from mammalian cells, the use, and the technology used for production. Technology development for these products has centered around the differences in characteristics of mammalian versus microbial cells, notably, the shear sensitivity and susceptibiUty to contamination of the mammalian lines. 

Superoxide dismutase has been approved by the FDA for preventing reperfusion injury or damage to donor organ tissue (178). This enzyme is prepared by recombinant DNA technology and marketed by Bristol-Myers and Pharmacia-Chiron. 

Microbes for Industiial and Agiicultural Applications, Dekker, NY, 1993. Click, B. R. and J. J. Pasternak, Molecular Biotechnology Trinciples and Applications of Recombinant DNA, ASM Press, Herndon, VA, 1994. Bajpai, Rakesb K., and Ales Prokop, eds. Recombinant DNA Technology II, Annals of the New York Academy of Sciences, vol. 721, 1993. 

Polymerase Chain Reacdon (PCR) Recombinant DNA Technology An Excidng Sciendfic Frontier... 

Recombinant DNA technology now verges on the ability to engineer at will the genetic constitution of organisms for desired ends. The commercial production of therapeutic biomolecules in microbial cultures is already established (for example, the production of human insulin in quantity in E. coli cells). Agricultural crops with desired attributes, such as enhanced resistance to her-... 

Baldwin, T. O., etal. (1987). Structural studies of bacterial luciferases results from recombinant DNA technology. In Schoelmerich, J. (ed.), Biolumin. Chemilumin., Proc. Int. Biolumin. Chemilumin. Symp., 4th, 1986, pp. 351-360. Wiley, Chichester, UK. 

Sakaki, Y., et al. (1988). Structure and function of the calcium-binding photoprotein aequorin studies by recombinant DNA technology. In Yagi, Y., and Miyazaki, T. (eds.), Calcium Signal Cell Response, pp. 151-156. Jpn. Sci. Soc. Press Tokyo, Japan. 

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