The Science:
Making DNA in a tube
When they published the structure of DNA in 1953, Francis Crick and James Watson (Nobel Laureates in Medicine, 1962) described the double helix as lending itself easily to a mechanism for its replication. Subsequent years of study proved their hypothesis, but it took decades for scientists to harness this power in the lab.
Until the mid-1980s, scientists painstakingly sequenced genes of interest using chemical methods that were difficult to perform and not suitable for long stretches of DNA such as entire genes, let alone an entire genome. Scientists also wanted to be able to chemically synthesize DNA in the laboratory, rather than isolating it from cells, although this process also proved difficult.
The cell, however, has no problem conducting either of these tasks, as enzymes called DNA polymerases perform this function. They “melt” DNA into its two complementary strands and chug along the chromosome to faithfully replicate each strand, giving rise to a duplicate genome that can produce two daughter cells in cell replication.
Double-stranded DNA is a perfect complement of hydrogen-bonded nucleotide bases and repetitive units of a sugar bonded to a nitrogenous base. The four bases that compose DNA are adenine (A), guanine (G), cytosine (C), and thymine (T), which differ only at the nucleotide base. A single strand of DNA is able to bind to its complementary strand through hydrogen-bonding: A’s bind to T’s, and G’s with C’s. It is this arrangement that lends itself so well to replication. Opening up the double strand creates two templates, each one capable of copying a new, complementary strand. It is this process that scientists had been trying to replicate in the test tube.
Kary Mullis became interested in the chemical synthesis of DNA in the lab after attending a lecture describing a success story. He hit the library to learn all that he could about DNA synthesis, and got a job at Cetus in Southern California developing DNA synthesis. Cetus became very good at making small DNA molecules for biologists, yet synthesizing long stretches of DNA still eluded them.
Mullis had his ground-breaking first ideas about PCR one night while driving to his cabin along the Pacific Coast Highway. No one at Cetus shared his enthusiasm. Undaunted, Mullis performed his first successful PCR reaction in December 1983 by amplifying a short bacterial sequence.
PCR has three main steps: melting, annealing, and extension. In the first step, the PCR mixture of template DNA, a short strand of DNA called a primer, free DNA bases, and polymerase enzyme are heated to 95 degrees Celsius. At this temperature, the double-stranded template DNA is “melted” into single-stranded DNA. The following annealing step is a cooling step to a low temperature at which DNA strands will begin binding with one another again. The primer DNA (that is complementary to only one site on the template DNA) can seek out its binding site and anneal to it.
The next step is where the magic happens: the sample is heated to 72 degrees Celsius and the DNA polymerase starts to work, extending the length of the primer one base at a time making a new strand complementary to its template. These three steps can be repeated hundreds of times to produce enough DNA material.
Mullis summarized the process: “beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat.”
This simple protocol has revolutionized biology. It has changed the way crimes are investigated, allowed us to analyze the genetic basis for diseases, developed the field of paleobiology, and resulted in the sequencing of the human genome, as well as those of countless other species.
Playing with proteins
The cell uses proteins to perform a multitude of functions, such as building the cell architecture. Proteins also act as enzymes and hormones. For decades, scientists have known that single-point mutations in a protein sequence can have dramatic effects on the function and stability of that protein. Many human diseases, such as cystic fibrosis and sickle cell anemia, are caused by a single mutation in a single protein. Scientists are therefore interested in studying these effects in the lab by producing point-mutant proteins to compare to the normal protein’s functions.
This type of work was not possible before Michael Smith developed site-directed mutagenesis. Until then, scientists had to rely on studying only naturally-occurring mutations and could not design new ones themselves.
Smith’s insight came while working at the University of British Columbia in 1978. Site-directed mutagenesis involves using a short piece of DNA (like those used as primers in PCR) that is not fully complementary to its target site. The piece of DNA contains the mutated DNA base(s) that will convert a normal protein coding sequence into the desired point-substituted sequence. This short piece of DNA can be injected into a target organism and at the next replication event, one of the daughter cells will contain the engineered mutation. Once the genome has the desired mutation, the protein that the cell makes from the newly mutated gene will also be changed.
Using Smith’s site-directed mutagenesis, scientists can create point substitutions, deletions, or insertions to a particular protein sequence, thus removing part of a protein, or adding a new sequence to it.
Today this process is made even simpler by employing PCR: the mutation is encoded in a primer and the reaction can take place in a test tube rather than in the host organism.
Most of the implications of site-directed mutagenesis are limited to molecular biology labs—it is used as a method to create mutations with which to test biological problems—but some have real world applications as well. For instance, the mutagenesis of an enzyme that is commonly added to laundry detergent has allowed the enzyme to perform better at low temperatures, allowing detergent to be more efficient in cold water.
What you may not know:
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Mullis is a controversial figure. He is venerated by some as a modern-day genius unafraid to go against dogma, but derided by others as an arrogant ego-maniac. He has been open about his recreational drug use (claiming PCR would not have been invented without LSD), and his alleged abduction by aliens (they looked like raccoons). He is also the first Nobel Laureate surfer. There is also much controversy over who actually invented PCR, as other scientists have earlier, credible claims to it.
B-students rejoice!
Smith was a mediocre student, completing an undergraduate and PhD degree in chemistry at the University of Manchester. After his graduate degree, Smith applied to many West Coast American Schools for post-doctoral training and was rejected from each one.
Smith did not let this rejection stand in his way. He eventually accepted a position at the laboratory of H. Gobind Khorana (Nobel Laureate in Medicine, 1968), who was new to the faculty at UBC. It is at UBC where Smith cut his teeth on biological chemistry and found his academic home. Smith died in Vancouver in 2000.
