Detailed studies of the structure and function of a gene at the molecular level require large quantities of the individual gene in pure form. A variety of techniques, often referred to as recombinant DNA technology, are used in DNA cloning, which permits researchers to prepare large numbers of identical DNA molecules. Recombinant DNA is simply any DNA molecule composed of sequences derived from different sources.

The key to cloning a DNA fragment of interest is to link it to a vector DNA molecule that can replicate within a host cell. After a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is replicated along with the vector, generating a large number of identical DNA molecules. The basic scheme can be summarized as follows:

Vector + DNA fragment

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Recombinant DNA

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Replication of recombinant DNA within host cells

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Isolation, sequencing, and manipulation of purified DNA fragment

Although investigators have devised numerous experimental variations, this flow diagram indicates the essential steps in DNA cloning.

In this section, we first describe methods for isolating a specific sequence of DNA from a sea of other DNA sequences. This process often involves cutting the genome into fragments and then placing each fragment in a vector so that the entire collection can be propagated as recombinant molecules in separate host cells. While many different types of vectors exist, our discussion will mainly focus on plasmid vectors in E. coli host cells, which are commonly used. Various techniques can then be employed to identify the sequence of interest from this collection of DNA fragments. Once a specific DNA fragment is isolated, the exact sequence of nucleotides in the fragment can be determined. We end with a discussion of the polymerase chain reaction (PCR). This powerful and versatile technique can be used in many ways to generate large quantities of a specific sequence and to manipulate DNA in the laboratory. The various uses of cloned DNA fragments are discussed in subsequent sections.