A particularly useful plasmid vector is based on the naturally occurring F plasmid of E. coli. Unlike smaller bacterial plasmids, the F plasmid—and its engineered derivative, the bacterial artificial chromosome (BAC)—is present in only one or two copies per E. coli cell. The fact that BACs are kept in such low numbers means that they can stably maintain very long DNA sequences, up to 1 million nucleotide pairs in length. With only a few BACs present per bacterium, it is less likely that the cloned DNA fragments will become scrambled by recombination with sequences carried on other copies of the plasmid. Because of their stability, ability to accept large DNA inserts, and ease of handling, BACs are now the preferred vector for handling large fragments of foreign DNA. As we will see below, BACs were instrumental in determining the complete nucleotide sequence of the human genome.
Often it is useful to break up a genome into much smaller fragments and clone every fragment, separately, using a plasmid vector. This approach is useful because it allows scientists to work with easily managed, discrete pieces of a genome instead of whole, unwieldy chromosomes.
This strategy involves cleaving genomic DNA into small pieces using a restriction nuclease (or, in some cases, by mechanically shearing the DNA) and ligating the entire collection of DNA fragments into plasmid vectors, using conditions that favor the insertion of a single DNA fragment into each plasmid molecule. These recombinant plasmids are then introduced into E. coli at a concentration that ensures that no more than one plasmid molecule is taken up by each bacterium. The collection of cloned plasmid molecules is known as a DNA library. Because the DNA fragments were derived directly from the chromosomal DNA of the organism of interest, the resulting collection—called a genomic library—will represent the entire genome of that organism (Figure 8–30), spread out over tens of thousands of individual bacterial colonies.
An alternative strategy, one that enriches for protein-coding genes, is to begin the cloning process by selecting only those DNA sequences that are transcribed into mRNA and thus correspond to protein-encoding genes. This is done by extracting the mRNA from cells and then making a DNA copy of each mRNA
Figure 8–30 Human genomic libraries containing DNA fragments that represent the whole human genome can be constructed using restriction nucleases and DNA ligase. Such a genomic library consists of a set of bacteria, each carrying a different fragment of human DNA. For simplicity, only the colored DNA fragments are shown in the library; in reality, all of the different gray fragments will also be represented.
Figure 8–29 A DNA fragment can be replicated inside a bacterial cell. To clone a particular fragment of DNA, it is first inserted into a plasmid vector, as shown in Figure 8–27. The resulting recombinant plasmid DNA is then introduced into a bacterium, where it is replicated many millions of times as the bacterium multiplies. For simplicity, the genome of the bacterial cell is not shown.
millions of genomic DNA fragments molecule present—a so-called complementary DNA, or cDNA. The copying reaction is catalyzed by the reverse transcriptase enzyme of retroviruses, which synthesizes a complementary DNA chain on an RNA template. The single-stranded cDNA molecules synthesized by the reverse transcriptase are converted by DNA polymerase into double-stranded cDNA molecules, and these molecules are inserted into a plasmid or virus vector and cloned (Figure 8–31). Each clone obtained in this way is called a cDNA clone, and the entire collection of clones derived from one mRNA preparation constitutes a cDNA library.
Figure 8–32 illustrates some important differences between genomic DNA clones and cDNA clones. Genomic clones represent a random sample of all of the DNA sequences in an organism—both coding and noncoding—and, with very rare exceptions, are the same regardless of the cell type used to prepare them. By contrast, cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library.