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The building blocks of all forms of life are cells. Simple organisms such as bacteria exist as single cells. Plants and animals are composed of many cell types, each organized into tissues and organs of specific functions. The determinants of genetic traits of living organisms are contained within the nucleus of each cell, in the form of a type of nucleic acids, called deoxyribonucleic acid (DNA). The genetic information in DNA is used for the synthesis of proteins unique to a cell. The ability of cells to express information coded by DNA in the form of protein molecules is achieved by a two-stage process of transcription and translation.

What is the chemical structure of a deoxyribonucleic acid (DNA) molecule? DNA is a polymer of deoxyribonucleotides. All nucleic acids consist of nucleotides as building units. A nucleotide has three components: sugar, base, and a phosphate group. (The combination of a sugar and a base is a nucleoside.) In the case of DNA, the nucleotide is known as deoxyribonucleotide, because the sugar in this case is deoxyribose. The base is either a purine (adenosine or guanine) or a pyrimidine (thymine or cytosine) (Fig. 2.1). Another type of nucleic acid is ribonucleic acid (RNA), a polymer of ribonucleotides also consisting of three components - a sugar, a base and a phosphate, except that the sugar in this case is a ribose, and that the base thymine is replaced by uracil.

The gene products from transcription and translation are proteins. The structure, and hence the functional property of a particular protein are specified by the information in its gene. Some understanding of the molecular structure of proteins is needed to make sense of the genetic processes.

Two processes are central to genetic continuity from one generation to the next: (1) Genetic information is conveyed from DNA to RNA to proteins (transcription and translation); (2) Genetic information is transferred from DNA to DNA (replication).

A gene is a discrete segment of DNA existing as an expression unit (see Section 1.1). A DNA molecule may consist of many genes. For example, in bacteriophage λ, all the genetic information is stored in a single DNA molecule with 48.5 kb, consisting of 60 genes. In humans, the genetic information is stored in 46 DNA molecules organized as 23 pairs of chromosomes, amounting to a total of 3.2 × 10 bp, and estimated ∼31,000 genes (see Section 1.4).

In this Chapter, we will apply what have been learned in the previous chapters to read the nucleotide sequences of a prokaryotic and a eukaryotic gene. The aim is to provide a simple guide for reading a gene sequence. Starting with a DNA sequence, one can extract a wealth of information on the architectural organization of the gene, including many of the features at the protein level. Both transcriptional and translation processes can be inferred by reading a gene sequence (Refer to previous chapters for basic concepts).

The manipulation of DNA utilizes a number of enzymes. These enzymes are naturally occurring in cells involved in transcription, translation, replication and other biological processes. The reactions catalyzed by these enzymes have become an essential part of gene cloning. Examples of enzyme uses in cloning include cutting and joining DNA, deletion or extension of DNA, generating new DNA fragments, and copying DNA from RNA. These enzymes are available commercially in highly purified forms suitable for cloning work.

The theoretical and experimental background for the cloning techniques is closely tied with the biological processes described in Part 1. Volumes of protocols are available for use in gene cloning. Fortunately, the fundamentals of the techniques involved are not difficult to understand.

The introduction of a foreign DNA into a host cell in many cases requires the use of a vector. Vectors are DNA molecules used to transfer a gene into a host (microbial, plant, animal) cell, and to provide control elements for replication and expression. The vector to be used is determined by the type of host cells and the objectives of the cloning experiment.

After insertion of a foreign DNA into a vector, the next step is to introduce the resulting recombinant DNA into a suitable host cell. The process of introducing DNA into living cells is called transformation. In a special case where the introduced DNA is a viral DNA, the process is called transfection. The choice of methods depends on the type of host systems in use, as well as the objective of cloning. Some of the procedures for transformation have been briefly mentioned in the discussion of vectors. A more detailed discussion on this important process is presented in this chapter.