This sheep, Dolly, was the first animal to be cloned from a single adult cell. The lamb you see beside her is her offspring, normal in every respect. From Dolly we learn that genes are not lost during development. If a single adult cell can be induced to switch the proper combination of genes on and off, that one cell can develop into a normal adult individual. Embryonic stem cells are like this—poised to become any cell of the body as the embryo develops. It may be possible to replace damaged tissues with healthy tissue grown from a patient’s own embryonic stem cells, so long as the disorder is not an inherited one. The approach has been used successfully in laboratory mice to cure a variety of disorders. However, its use in humans would require employing stem cells derived from the patient, and because this may involve destroying a human embryo the approach is controversial. Another approach, when the damaged tissue does result from a defective gene, is to repair rather than replace, using a virus to transfer a healthy gene into those tissues that lack it. In this chapter you will explore genomic screening, the application of gene technology to medicine and agriculture, reproductive cloning, stem cell tissue replacement, and gene therapy, all areas in which a revolution is reshaping biology.

13.1. Genomics

Recent years have seen an explosion of interest in comparing the entire DNA content of different organisms, a new field of biology called genomics. While initial successes focused on organisms with relatively small numbers of genes, researchers have recently completed the sequencing of several large eukaryotic genomes, including our own.

The full complement of genetic information of an organism—all of its genes and other DNA—is called its genome. To study a genome, the DNA is first sequenced, a process that allows each nucleotide of a DNA strand to be read in order. The first genome to be sequenced was a very simple one: a small bacterial virus called Ø-X174 (Ø is the Greek letter phi). Frederick Sanger, inventor of the first practical way to sequence DNA, obtained the sequence of this 5,375-nucleotide genome in 1977. This was followed by the sequencing of dozens of prokaryotic genomes. The advent of automated DNA sequencing machines in recent years has made the DNA sequencing of much larger eukaryotic genomes practical, including our own (table 13.1).

TABLE 13.1. SOME EUKARYOTIC GENOMES

Sequencing DNA

In sequencing DNA, the DNA of unknown sequence is first cut into fragments. Each DNA fragment is then copied (amplified), so there are thousands of copies of the fragment. The DNA fragments are then mixed with copies of DNA polymerase, copies of a primer (recall from chapter 11 that DNA polymerase can only add nucleotides onto an existing strand of nucleotides), a supply of the four nucleotide bases, and a supply of four different chain-terminating chemical tags. The chemical tags act as one of the four nucleotide bases in DNA synthesis, undergoing complementary base pairing. First, heat is applied to denature the double-stranded DNA fragments. The solution is then allowed to cool, allowing the primer (the lighter blue box in figure 13.1 1) to bind to a single strand of the DNA, and synthesis of the complementary strand proceeds. Whenever a chemical tag is added instead of a nucleotide base, the synthesis stops, as shown in the figure. For example, the terminating red “T” was added after three normal nucleotides and synthesis stopped. Because of the relatively low concentration of the chemical tags compared with the nucleotides, a tag that binds to G on the DNA fragment, for example, will not necessarily be added to the first G site. Thus, the mixture will contain a series of double-stranded DNA fragments of different lengths, corresponding to the different distances the polymerase traveled from the primer before a chain-terminating tag was incorporated (six are shown in 1).

Figure 13.1. How to sequence DNA.

1 DNA is sequenced by adding complementary bases to a single-stranded fragment. DNA synthesis stops when a chemical tag is inserted instead of a nucleotide, resulting in different sizes of DNA fragments. 2 The DNA fragments of varying lengths are separated by gel electrophoresis, the smaller fragments migrating farther down the gel. (The boldface letters indicate the chemical tags added in step 1 that stopped the replication process.) 3 Computers scan the gel, from smallest to largest fragments, and display the DNA sequence as a series of colored peaks. 4 Data from an automated DNA-sequencing run show the nucleotide sequence for a small section of the Arabidopsis (plant) genome.

The series of fragments are then separated according to size by gel electrophoresis. The fragments become arrayed like the rungs of a ladder, each rung being one base longer than the one below it. Compare the lengths of the fragments in 1 and their positions on the gel in 2. The shortest fragment has only one nucleotide (G) added to the primer, so it is the lowest rung on the gel. In automated DNA sequencing, fluorescently colored chemical tags are used to label the fragments, one color for each type of nucleotide. Computers read the colors on the gel to determine the DNA sequence and display this sequence as a series of colored peaks (3 and 4). The development of automated sequencers in the mid-1990s has allowed the sequencing of large eukaryotic genomes. A research institute with several hundred such instruments can sequence 100 million base pairs every day, with only 15 minutes of human attention!

Key Learning Outcome 13.1. Powerful automated DNA sequencing technology is now revealing the DNA sequences of entire genomes.