The discovery and understanding of the degenerate genetic code, with its sequences or codons, led to the possibility of changing and manipulating DNA, with the discovery of restriction enzymes in bacteria that could be used to "cut and paste" DNA sequences.
In 1983 Kary Mullis, an American biochemist, developed the Polymerase Chain Reaction method of amplifying small amounts of DNA; although the main principles of PCR were first described in 1971 by Kjell Kleppe, a Norwegian scientist. PCR is now used in many genetic techniques requiring the amplification of DNA, including genetic fingerprinting, paternity testing, diagnosis of genetic disorders and the analysis of ancient DNA. As time progressed throughout the twentieth century, further uses for PCR were discovered, leading to the introduction of gene therapy, genetically modified crops, cloning of genes and use of stem cells.
Furthermore, in the late 20th century the task became to sequence particular genes (for example, the gene in which defects cause cystic fibrosis) and map the entire genomes of increasingly complex organisms. The Human Genome Project was completed in 2003.
Polymerase Chain Reaction Technique
The polymerase chain reaction serves to duplicate DNA. It uses many repeated cycles, each of which consists of three steps:
1. The reaction solution is heated to 95°C. This reaction solution contains the DNA molecules to be copied, polymerases which copy the DNA, primers (which serve as starting DNA) and nucleotides which are attached to the primers. This heating to a high temperature causes the two complementary strands to separate via denaturation or melting.
2. Next, the temperature is lowered to 55°C, which causes the primers to bind to the DNA, a process known as hybridisation or annealing. The resulting bonds are stable only if the primer and DNA segment are complementary, i.e. if the base pairs of the primer and DNA segment match. The polymerases then commence attaching further complementary nucleotides at these sites, thus strengthening the bonding between the primers and the DNA.
3. The third step is extension, where the temperature is again increased, this time to 72°C. This is the optimum working temperature for the polymerases used, which add further nucleotides to the developing DNA strand. At the same time, any loose bonds which have formed between the primers and DNA segments that are not fully complementary are broken. Each time these three steps are repeated the number of duplicate DNA molecules is doubled. After 20 cycles, approximately one million molecules are cloned from a single segment of double stranded DNA.
The temperatures and duration of the individual steps described above refer to the most commonly used protocol.
One of the primary modifications of the original protocol pertained to the polymerases used.
In common with all enzymes, polymerases function best at the body temperature of the organism in which they originate - 37°C in the case of polymerases isolated from humans. Below this temperature, the enzyme's activity declines steeply, above this temperature, it is quickly destroyed. In PCR, however, the two strands of the DNA molecule must be separated in order to allow the primers to anneal to them. This is achieved by increasing the temperature to around 95°C. At such elevated temperatures, the polymerases of the vast majority of organisms are permanently denatured and destroyed.
Hot springs yielded the solution. Certain microorganisms thrive in such environments under the most hostile conditions, at temperatures that can reach 100°C and in some cases, in the presence of excessive salt and/or acid concentrations. The polymerases of these organisms are biologically adapted to high temperatures and are therefore ideal for use in PCR. Now, the polymerases used in nearly all PCR methods throughout the world are derived from such microorganisms.
This exceptionally hardy bacterium is known as Thermus aquaticus, and its heat-stable polymerase, called Taq polymerase, supports an entire biochemical industry. The organism was originally discovered in a 70°C spring in Yellowstone National Park in the USA. Employees of Cetus, who Kary Mullis was working for at the time of his discovery, isolated the first bacterial samples from the hot spring and then cultivated in the laboratory one of the most scientifically valuable bacterial strains known to the world today. Meanwhile, Thermus aquaticus has since been discovered in similar hot springs all over the world.
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