tissue (e.g., brain) double-stranded cDNA copy of original mRNA
Figure 8–31 The synthesis of cDNA. Total mRNA is extracted from a particular tissue, and the enzyme reverse transcriptase (see Figure 5–62) is used to produce DNA copies (cDNA) of the mRNA molecules. For simplicity, the copying of just one of these mRNAs into cDNA is illustrated. A short oligonucleotide complementary to the poly-A tail at the 3ʹ end of the mRNA (discussed in Chapter 6) is first hybridized to the RNA to act as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA–RNA hybrid helix. Treating the DNA–RNA hybrid with a specialized nuclease (RNAse H) that attacks only the RNA produces nicks and gaps in the RNA strand. DNA polymerase then copies the remaining single-stranded cDNA into double-stranded cDNA. Because DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base-paired to the 3ʹ end of the first DNA strand usually acts as the primer for the second strand synthesis, as shown. Any remaining RNA is eventually degraded during subsequent cloning steps. As a result, the nucleotide sequences at the extreme 5ʹ ends of the original mRNA molecules are often absent from cDNA libraries.
Genomic libraries are especially useful in determining the nucleotide sequences of a whole genome. For example, to determine the nucleotide sequence of the human genome, it was broken up into roughly 100,000-nucleotide-pair pieces, each of which was inserted into a BAC plasmid and amplified in E. coli. The resulting genomic library consisted of tens of thousands of bacterial colonies, each containing a different human DNA insert. The nucleotide sequence of each insert was determined separately and the sequence of the entire genome was stitched together from the pieces.
The most important advantage of cDNA clones, over genomic clones, is that they contain the uninterrupted coding sequence of a gene. When the aim of the cloning, for example, is to produce the protein in large quantities by expressing the cloned gene in a bacterial or yeast cell, it is more preferable to start with cDNA.
Genomic and cDNA libraries are inexhaustible resources, which are widely shared among investigators. Today, many such libraries are also available from commercial sources. Because the identity of each insert in a library is often known (through sequencing the insert), it is often possible to order a particular region of a chromosome (or, in the case of cDNA, a complete, intron-less protein-coding gene) and have it delivered by mail.
Cloning DNA by using bacteria revolutionized the study of genomes and is still in wide use today. However, there is an even simpler way to clone DNA, one that can be carried out entirely in vitro. We discuss this approach, called the polymerase chain reaction, below. However, first we need to review a fundamental, far-reaching property of DNA and RNA called hybridization.
Figure 8–32 The differences between cDNA clones and genomic DNA clones derived from the same region of DNA.
In this example, gene A is infrequently transcribed, whereas gene B is frequently transcribed, and both genes contain introns (orange). In the genomic DNA library, both the introns and the nontranscribed DNA (gray) are included in the clones, and most clones contain, at most, only part of the coding sequence of a gene (red). In the cDNA clones, the intron sequences (yellow) have been removed by RNA splicing during the formation of the mRNA (blue), and a continuous coding sequence is therefore present in each clone. Because gene B is transcribed more frequently than gene A in the cells from which the cDNA library was made, it is represented much more frequently than A in the cDNA library. In contrast, A and B are represented equally in the genomic DNA library.
Hybridization Provides a Powerful, But Simple Way to Detect Specific Nucleotide Sequences