Recombinant DNA Constructs

Vectors—vehicles for introducing recombinant-DNA constructs into recipient organisms or cells, such as plasmids, viruses, or a bacteria. 

Figure 18 A illustrates the basic features of recombinant DNA construction. The process begins by using a restriction enzyme (Biochemical Methods 17.1) that generates sticky ends to cleave DNA from two different sources. The DNA fragments are then mixed under conditions that allow annealing (base pairing) between the sticky ends to occur. Once base pairing has occurred, the fragments are covalently bonded together by DNA ligase. After recombinant DNA molecules...

Key words Overlap extension PCR, GC-rich annealing sequences. Recombinant DNA construction. 

This chapter describes the chemistry of nucleotides and the m or classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules. 

Fig. 24.3 The construction of a chimeric (or recombinant) DNA molecule hy joining together two DNA fragments produced by cleavage of different parental DNA molecules with the same restriction endonuclease.

Liu, Q Li, M. Z., Leibham, D., Cortez, D., and Elledge, S. J. (1998). The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Current Biology 8, 1300-1309. 

The advent of recombinant DNA technology led to the development of antibodies and fragments that are tailored for optimal behaviour in vivo [7,8]. Humanized and chimeric antibodies can be constructed to circumvent the human anti-mouse antibody response elicited by mouse antibody treatment of patients, which severely hampers the application of these powerful molecules. The treatment of rheumatoid arthritis patients with doses of as high as 10 mg kg cA2 chimeric antibody specific for TNFa [9], emphasizes that at present the production and purification methods for these proteins have been optimized to such extent that clinical studies can be considerably intensified. 

Development of Proteinaceous Drug Targeting Constructs Using Chemical and Recombinant DNA Approaches... 

By using recombinant DNA techniques, modifications in the protein backbone, such as additions, deletions and alterations of amino acids, are easily achieved. These modifications can contribute to improved pharmacokinetic properties of the construct. Additions may consist of the introduction of residues that allow covalent conjugation of drug molecules. Deletions of amino acids can employed to remove membrane-bound regions of a protein, thereby increasing its solubility. Single amino acid modifications can be used to minimize antibody responses and alter the binding specificity and/or the three-dimensional structure of a certain protein. 

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