American Nobel laureate in chemistry Kary Mullis has died in California at the age of 74. According to his wife, death occurred on August 7. The reason is heart and respiratory failure due to pneumonia.
About what contribution he made to biochemistry and for which he received the Nobel Prize, we will be told by James Watson himself, the discoverer of the DNA molecule.
An excerpt from a book by James Watson, Andrew Berry, Kevin Davis
DNA. History of the genetic revolution
Chapter 7. The human genome. life script
...
The polymerase chain reaction (PCR) was invented in 1983 by biochemist Cary Mullis, who worked for Cetus. The discovery of this reaction was quite remarkable. Mullis later recalled: βOne Friday night in April 1983, I had a flash of light. I was driving down a moonlit winding mountain road into Northern California, the edge of the redwoods.β It is impressive that it was in this situation that he was visited by inspiration. And it's not at all that northern California has special roads that contribute to insight; it's just that a friend of his once saw Mullis recklessly rushing down an icy two-way road and it didn't bother him at all. A friend told the New York Times: βMullis imagined he was going to die when he crashed into a redwood tree. Therefore, he is not afraid of anything while driving, if there are no redwoods along the road. The presence of redwoods along the road forced Mullis to focus and ... here it is, insight. For his invention in 1993, Mullis received the Nobel Prize in Chemistry and since then has become even more strange in his actions. For example, he is a supporter of the revisionist theory that AIDS is not related to HIV, which significantly undermined his own reputation and interfered with doctors.
PCR is a fairly simple reaction. To carry it out, we need two chemically synthesized primers that are complementary to opposite ends of different strands of the required DNA fragment. Primers are short stretches of single-stranded DNA, each about 20 base pairs long. The peculiarity of the primers is that they correspond to the sections of DNA that need to be amplified, that is, the DNA template.
(Image clickable) Carey Mullis, inventor of PCR
The specificity of PCR is based on the formation of complementary complexes between the template and primers, short synthetic oligonucleotides. Each of the primers is complementary to one of the chains of the double-stranded template and limits the beginning and end of the amplified region. In fact, the obtained βmatrixβ is a whole genome, and our goal is to isolate fragments of interest from it. To do this, the double-stranded DNA template is heated to 95 Β°C for several minutes to separate the DNA strands. This step is called denaturation because the hydrogen bonds between the two strands of DNA are broken. When the strands are separated, the temperature is lowered to allow the primers to bind to the single stranded template. DNA polymerase begins DNA replication by binding to a segment of a chain of nucleotides. The DNA polymerase enzyme replicates the template strand using a primer as a primer or copy template. As a result of the first cycle, we obtain multiple sequential doubling of a certain section of DNA. Next, we repeat this procedure. After each cycle, we obtain a target site in double quantity. After twenty-five PCR cycles (that is, in less than two hours), we have the DNA section of interest to us in an amount 225 times greater than the original (that is, we amplified it about 34 million times). In fact, at the input we got a mixture of primers, template DNA, DNA polymerase enzyme and free bases A, C, G and T, the amount of a specific reaction product (limited by primers) grows exponentially, and the number of βlongβ DNA copies is linear, therefore, in reaction products dominates.
Amplification of the desired DNA region: polymerase chain reaction
In the early days of PCR, the main problem was that after each heating-cooling cycle, DNA polymerase had to be added to the reaction mixture, since it was inactivated at 95 Β°C. Therefore, it was necessary to re-add it before each of the 25 cycles. The reaction procedure was relatively inefficient, required a lot of time and the polymerase enzyme, and the material is very expensive. Luckily, Mother Nature came to the rescue. Many animals feel comfortable at temperatures well above 37Β°C. Why is 37Β°C so important to us? This is because this temperature is optimal for E. coli, from which the polymerase enzyme for PCR was originally obtained. In nature, there are microorganisms whose proteins, over millions of years of natural selection, have become more resistant to high temperatures. It has been proposed to use DNA polymerases from thermophilic bacteria. These enzymes proved to be thermostable and were able to withstand many reaction cycles. Their use made it possible to simplify and automate PCR. One of the first thermostable DNA polymerases was isolated from the bacterium Thermus aquaticus, which lives in the hot springs of Yellowstone National Park, and was named Taq polymerase.
PCR quickly became the main workhorse of the Human Genome Project. In general, the process does not differ from that developed by Mullis, it was just automated. We no longer depended on a crowd of blind-eyed graduate students painstakingly pouring droplets of liquid into plastic test tubes. In modern laboratories that carry out molecular genetic research, this work is performed on robotic conveyors. The PCR robots involved in a sequencing project as massive as the Human Genome are working relentlessly with huge volumes of heat-resistant polymerase. Some scientists working in the Human Genome Project were outraged by the unreasonably high fees that the owner of the PCR patent, the European industrial and pharmaceutical giant Hoffmann-LaRoche, adds to the cost of consumables.
Another βdriving startβ was the DNA sequencing method itself. The chemistry behind this method was no longer a novelty at the time: The Human Genome Project (HGP) had adopted the same ingenious method developed by Fred Senger back in the mid-1970s. The innovation was in the scale and degree of automation that sequencing could achieve.
Automated sequencing was originally developed in Lee Hood's lab at Caltech. He graduated from high school in Montana and played American football as a forward; thanks to Hood, the team won the state championship more than once. Team interaction skills were useful to him in his scientific career. Hood's lab housed a motley crew of chemists, biologists, and engineers, and his lab soon became a leader in technological innovation.
In fact, the automatic sequencing method was invented by Lloyd Smith and Mike Hunkapiller. Mike Hunkapiller, then working in Hood's lab, approached Lloyd Smith with an improved sequencing method that would stain each type of base a different color. Such an idea could quadruple the efficiency of the Sanger process. Sanger's sequencing in each of the four test tubes (according to the number of bases) with the participation of DNA polymerase produces a unique set of oligonucleotides of different lengths, including the primer sequence. Next, formamide was added to the tubes to separate the chains, and electrophoresis was performed in a polyacrylamide gel in four lanes. In the Smith and Hunkapiller variant, dideoxynucleotides are labeled with four different dyes and PCR is performed in one tube. Then, during polyacrylamide gel electrophoresis, a laser beam at a specific location in the gel excites the activity of the dyes, and the detector determines which nucleotide is currently migrating through the gel. At first, Smith was pessimistic - he was afraid that the use of ultra-low doses of the dye would lead to the fact that the nucleotide regions would be indistinguishable. However, having an excellent understanding of laser technology, he soon found a way out using special fluorochrome dyes that fluoresce when exposed to laser radiation.
(Full version - 4,08 MB) Small print: DNA sequence decoded using an automatic sequencer, obtained from an automatic sequencing machine. Each color corresponds to one of the four bases
In the classical version of the Sanger method, one of the strands of the analyzed DNA acts as a template for the synthesis of a complementary strand by the DNA polymerase enzyme, then the sequence of DNA fragments is sorted by size in the gel. Each fragment, which is included in the DNA during synthesis and allows subsequent visualization of the reaction products, is labeled with a fluorescent dye corresponding to the terminal base (this was discussed on p. 124); therefore, the fluorescence of this fragment will be an identifier for this base. Then it remains only to carry out the detection and visualization of the reaction products. The results are analyzed by computer and presented as a sequence of colored peaks corresponding to four nucleotides. The information is then transferred directly to the computer's information system, which eliminates the time-consuming and sometimes painful data entry process that made sequencing very difficult.
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Source: habr.com