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THE SECRETS OF LIFE: A MATHEMATICIAN'S INTRODUCTION TO MOLECULAR BIOLOGY 13 THE RECOMBINANT DNA REVOLUTION By 1965, molecular biology had laid bare the basic secrets of life. Without the ability to manipulate genes, however, the understanding was more theoretical than operational. In the 1970s, this situation was transformed by the recombinant DNA revolution. Biochemists discovered a variety of enzymes made by bacteria that allowed one to manipulate DNA at will. Bacteria made restriction enzymes, which cut DNA at specific sequences and served as a defense against invading viruses, and ligases, which join DNA fragments. With these and other tools (which are now all readily available from commercial suppliers), it became possible to cut and paste DNA fragments at will and to introduce them into living cells (Figure 1.8). Such cloning experiments allow scientists to reproduce unlimited quantities of specific DNA molecules and have led to detailed understanding of individual genes. Moreover, producing recombinant DNA molecules that contain bacterial DNA instructions for making a particular human protein (such as insulin) gave birth to the biotechnology industry. A key development was the invention of DNA sequencing, the process of determining the precise nucleotide sequence of a cloned DNA molecule. With DNA sequencing, it became possible to read the sequence of any gene in stretches of 300 to 500 nucleotides at a time. DNA sequencing has revealed striking similarities among living creatures as diverse as humans and yeast, with far-reaching consequences for our understanding of molecular structure and evolution. DNA sequencing has also led to an information explosion in biology, with public databases still expanding at a rapid exponential rate. In early 1993, there were over 100 million bases of DNA in the public databases. For reference, the entire genome of the intestinal bacteria Escherichia coli (E. coli) consists of about 4.6 million bases, and the human genome sequence has roughly 3 billion bases. In recent years a powerful new technique called the polymerase chain reaction (PCR) has been added to the molecular biologist's tool kit (Figure 1.9). PCR allows one to directly amplify a specific DNA sequence without resort to cloning. To perform PCR, one uses short DNA molecules called primers (typically about 20 bases long) that are complementary to the sequences flanking the region of interest. Each
THE SECRETS OF LIFE: A MATHEMATICIAN'S INTRODUCTION TO MOLECULAR BIOLOGY 14 Figure 1.8 By cloning a foreign DNA molecule in a plasmid vector, it is possible to propagate the DNA in a bacterial or other host cell.
THE SECRETS OF LIFE: A MATHEMATICIAN'S INTRODUCTION TO MOLECULAR BIOLOGY 15 Figure 1.9 The polymerase chain reaction (PCR) allows exponential amplification of DNA. The method involves successive rounds of copying (using the enzyme DNA polymerase) between two synthetic primers corresponding to nearby DNA sequences. Each round doubles the number of copies. Courtesy of the Perkin-Elmer Corporation. Reprinted from the National Research Council (1992).
