Sequence databases can be searched to find similar amino acid or nucleic acid sequences. Here, a search for proteins similar to the human cell-cycle regulatory protein Cdc2 (Query) locates maize Cdc2 (Sbjct), which is 68% identical to human Cdc2 in its amino acid sequence. The alignment begins at residue 57 of the Query protein, suggesting that the human protein has an N-terminal region that is absent from the maize protein. The green blocks indicate differences in sequence, and the yellow bar summarizes the similarities: when the two amino acid sequences are identical, the residue is shown; similar amino acid substitutions are indicated by a plus sign (+). Only one small gap has been introduced—indicated by the red arrow at position 194 in the Query sequence—to align the two sequences maximally. The alignment score (Score), which is expressed in two different types of units, takes into account penalties for substitutions and gaps; the higher the alignment score, the better the match. The significance of the alignment is reflected in the Expectation (E) value, which specifies how often a match this good would be expected to occur by chance. The lower the E value, the more significant the match; the extremely low value here (e–111) indicates certain significance. E values much higher than 0.1 are unlikely to reflect true relatedness. For example, an E value of 0.1 means there is a 1 in 10 likelihood that such a match would arise solely by chance.
analyze, DNA has become the easiest. It is now possible to determine the entire nucleotide sequence of a bacterial or fungal genome in a matter of hours, and the sequence of an individual human genome in less than a day. Once the nucleotide sequence of a genome is known, any individual gene can be easily isolated, and large quantities of the gene product (be it RNA or protein) can be made either by introducing the gene into bacteria or animal cells and coaxing these cells to over-express the foreign gene or by synthesizing the gene product in vitro. In this way, proteins and RNA molecules that might be present in only tiny amounts in living cells can be produced in large quantities for biochemical and structural analyses. And this approach can also be used to produce large quantities of human proteins (such as insulin, or interferon, or blood-clotting proteins) for use as human pharmaceuticals. As we will see later in this chapter, it is also possible for scientists to alter an isolated gene and transfer it back into the germ line of an animal or plant, so as to become a functional and heritable part of the organism’s genome. In this way, the biological roles of any gene can be assessed by observing—in the whole organism—the results of modifying it.
The ability to manipulate DNA with precision in a test tube or an organism, known as recombinant DNA technology has had a dramatic impact on all aspects of cell and molecular biology, allowing us to routinely study cells and their macromolecules in ways that were unimaginable even twenty years ago. Central to the technology are the following manipulations: 1.
Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual pieces of a genome.
2.
DNA ligation, which makes it possible to seamlessly join together DNA molecules from widely different sources.
3.
DNA cloning (through the use of either cloning vectors or the polymerase chain reaction) in which a portion of a genome (often an individual gene) is “purified” away from the remainder of the genome by repeatedly copying it to generate many billions of identical molecules.
4.
Nucleic acid hybridization, which makes it possible to identify any specific sequence of DNA or RNA with great accuracy and sensitivity based on its ability to selectively bind a complementary nucleic acid sequence.
5.
DNA synthesis, which makes it possible to chemically synthesize DNA molecules with any sequence of nucleotides, whether or not the sequence occurs in nature.
6.
Rapid determination of the sequence of nucleotides of any DNA or RNA molecule.
In the following sections, we describe each of these basic techniques which, together, have revolutionized the study of cell and molecular biology.