Welcome to the Genome. Michael Yudell
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the discovery of plasmids or what might be termed bacterial copying machines, other vehicles have been created that can copy larger pieces of DNA. The average limiting size of a plasmid is about 5000 bases. Phages, a specific class of viruses that infect bacteria and can be stably replicated by them, can carry about 15,000 bases; cosmids, an artificial cloning vector with a phage gene, can carry about 35,000 bases; bacterial artificial chromosomes (also known as BACs) can take over 100,000 bases of sequence; and yeast artificial chromosomes (also known as YACs) can take approximately 1,000,000. Although these microbial methods remain an important component of DNA sequencing and were central to the effort to sequence the human genome, they are all arduous ways to copy DNA. BAC‐copied DNA was used in the sequencing of the human genome. (29)
RESEARCH MILESTONE 6: SEQUENCING A VIRAL GENOME
By the 1970s advances in sequencing technology brought biology and genetics to the brink of the genomic revolution. The most important developments in sequencing technology occurred simultaneously in laboratories on opposite sides of the Atlantic. Two groups—biologist Walter Gilbert’s group at Harvard and Frederick Sanger’s group at Cambridge—exploited the chemistry of nucleic acids to come to the same brilliant idea. Unlike Edward Southern’s method, which revealed only the presence of DNA and genes, Gilbert’s and Sanger’s methods revealed the actual sequences of nucleotides along strands of DNA. The two methods had “complementary strengths,” and were used depending on what was to be sequenced. (30) The men shared the Nobel Prize in 1980 for this work. It was Sanger’s second Nobel. (31)
Sanger’s sequencing success rested on several premises. First, he knew that he could take a piece of DNA and synthesize its entire length with DNA polymerase. He was also aware of discoveries that showed that by using a class of nucleotides called chain terminators he could interrupt the synthesis of a DNA chain. These chain terminators come in four forms—terminator G, terminator C, terminator T, and terminator A—and when they were placed in a test tube with a DNA fragment and DNA polymerase and then placed on a gel, Sanger could determine the order of nucleotides in a given DNA fragment. He accomplished this by radioactively labeling the locations where the chain terminators stopped DNA synthesis at one of the four particular nucleotides. (32)
Sanger’s method of labeling fragments of DNA with radioactivity, using gel electrophoresis to separate the fragments, and using X‐ray film to visualize them quickly became commonplace in molecular biology laboratories and is still today the basis for gene sequencing. (33) In 1977, using his own method, Sanger himself accomplished the once unthinkable by completing the sequence of the entire genome of Phi‐X174, a virus that infects E. coli in the human digestive tract. Despite the fact that this virus was just over 5000 base pairs long, it took Sanger’s group years to sequence it. (34) By 2000 the Phi‐X174 genome could be sequenced in just a few hours.
The sequence itself revealed remarkable information about genes and gene structure. Among the most intriguing was the finding that even though there are 5386 nucleotides and nine proteins made from genes in the genome of Phi‐X174, calculations showed that there was not enough DNA to code for the proteins that the Phi‐X174 genome produced. This was confusing to scientists. The larger number of proteins than available DNA in PhiX was accounted for by some stretches of the genes in the PhiX genome coding for two or more different proteins by having one gene embedded in another. (35) This important finding is characteristic of many genomes, including the human genome. (36)
RESEARCH MILESTONE 7: THE ULTIMATE DNA COPYING TOOL
Few scientists have a moment of inspiration like the one that came to Kerry Mullis in 1983. According to Mullis, he was driving along a winding moonlit California mountain road when he thought up “a process that could make unlimited numbers of copies of genes.” As he drove, he designed the polymerase chain reaction (PCR) in his head. (37) PCR would soon become the newest and most advanced gene amplification technique, allowing for millions of copies of selected fragments of DNA to be made without plasmid cloning in as little as an hour, as opposed to the tedious vector‐based cloning that could take weeks or even months.
Mullis and his colleagues reasoned that four things were needed to make DNA: (i) a template (one of the strands of the target sequence from a double helix), (ii) the nucleotides (the basic building blocks of DNA—G, A, T, and C), (iii) primers (short single strands of DNA designed to find their base pair complements), and (iv) an enzyme, a DNA polymerase. They also recognized the key from previous work on DNA replication—that in order to replicate a specific region of DNA in a genome you would need to have two primers, one for each strand to be read in opposite directions. The distance between these primers would define the length of the sequence that this new method would amplify. (38)
With these basic tools and a simple but ingenious algorithm, Mullis created the three‐stage polymerase chain reaction. In the first step the temperature of DNA is raised to above 95–97 °C, a temperature at which the strands of a double helix come apart. Second, the temperature is lowered to 45–65 °C, which forces the primers to anneal or stick to the target region of DNA. Finally, at a temperature conducive to the DNA polymerase, the reaction is activated and replication begins.
These PCR cycles produce an astounding number of fragments between the two primers. It starts with only a single copy of a fragment. After one cycle of PCR two copies of the desired fragment exist. After two cycles four copies exist, and after three cycles eight copies exist. Four cycles make 16 copies. The increase in copy number is not linear, but rather geometric. Finally, after approximately 30 cycles, over a billion copies of a particular DNA segment will exist in the reaction.
Mullis had one serious problem to overcome. At 95 °C almost all cellular material denatures, destroying the needed polymerase in the PCR reaction. In the original PCR design, fresh polymerase had to be added after each cycle. By 1988, however, the cycle was modified by the addition of a DNA polymerase from the bacterium Thermus aquaticus, which normally thrives in and around deep‐water thermal vents and easily resists the 95 °C melting temperature in the PCR cycles. The cycle could thus run continuously without adding fresh polymerase by starting it at 94 °C (denaturing the DNA strands), lowering it to 45–65 °C (to anneal the primers), and then raising it to 72 °C (to activate the T. aquaticus, or Taq, polymerase). (39)
The molecular revolution was just over 30 years old by the mid‐1980s. Although so much had been accomplished since Watson and Crick’s groundbreaking discovery in 1953, the broader application of genetics was limited by the then‐current state of technology. Molecular biologists had established the basic physical and chemical rules of heredity, providing the biochemical tools to answer Schrodinger’s question What is Life? From Sanger’s basic sequencing tools, to the cracking of the genetic code, to the development of PCR, technologies were developed that brought science closer to answering Schrodinger’s question. But even with these tools scientists were only barely able to apply knowledge of cellular “life” to basic medical challenges. The genetics of sickle‐cell anemia, for example, have been understood for more than 50 years yet there is still no cure for this disease. The proposal to sequence the human genome in 1985 was an attempt to provide biology with something akin to chemistry’s periodic table. Such a catalog of the human genome, scientists hoped, would provide a foundation for improving our understanding of the relationship between genetics and human disease, and be a way to begin to apply nearly a century of work in genetics to health care. Much as Schrodinger’s question prompted a generation of scientists to investigate and uncover the molecular mechanisms of heredity, the sequencing of the human genome inspired scientists at the dawn of the twenty‐first century to develop a more precise and richer understanding of how our genomes work.
REFERENCES
1 1. Erwin Schrodinger. 1992. What is Life: The Physical Aspect of the Living Cell. New York: Cambridge University Press, p.5.
2 2. Schrodinger, 1992, p.6.
3 3. Schrodinger, 1992, p.3.
4 4. Schrodinger,