Recombinant DNA Techniques and Molecular Cloning

INTRODUCTION The human genome contains the blueprint for human development and maintenance, and may ultimately provide the means to understand human cellular and molecular processes in both health and disease. The genome is the full complement of DNA from an organism and carries all the information needed to specify the structure of every protein the cell can produce. The realisation that DNA lies behind all of the cell's activities led to the development of what is termed molecular biology. Rather than a discrete area of the biosciences, molecular biology is now accepted as a very important means of understanding and describing complex biological processes. The development of methods and techniques for studying processes at the molecular level has led to new and powerful ways of isolating, analysing, manipulating and exploiting nucleic acids. Moreover, to keep pace with the explosion in biological information, the discipline termed bioinformatics has evolved and provides a vital role in current biosciences. The completion of the human genome project and numerous other genome projects has allowed the continued development of new, exciting areas of biological sciences such as biotechnology, genome mapping, molecular medicine and gene therapy. In considering the potential utility of molecular biology techniques it is important to understand the basic structure of nucleic acids and gain an appreciation of how this dictates the function in vivo and in vitro. Indeed, many techniques used in molecular biology mimic in some way the natural functions of nucleic acids such as replication and transcription. This chapter is therefore intended to provide an overview of the general features of nucleic acid structure and function, and describe some of the basic methods of recombinant DNA technology, together with an overview of molecular cloning and associated methods. STRUCTURE OF NUCLEIC ACIDS Primary Structure of Nucleic Acids Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are macromolecular structures composed of regular repeating polymers formed from nucleotides . These are the basic building blocks of nucleic acids and are derived from nucleosides, which are composed of two elements: a five-membered pentose carbon sugar (2-deoxyribose in DNA and ribose in RNA) and a nitrogenous base.