Saturday, April 11, 2009
Thermus aquaticus
On page 70, Carroll writes "A heat-stable enzyme that could copy DNA at high temperatures was isolated from Thermus aquaticus. This enzyme led to the invention of a new, efficient, and very fast technique for the study of genes in any species. This technique catalyzed a vast expansion in the amount and diversity of DNA information that could be obtained from nature, as well as the creation of a multi-hundred-million-dollar market in DNA diagnostics and forensics." Discuss the impact of this enzyme and describe the technique created for the study of genes. What have we learned about DNA from this process? How is it used in DNA diagnostics and forensics? Use plenty of examples.
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The "heat-stable enzyme" referred to above is Taq polymerase. Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965 (Source: http://en.wikipedia.org/wiki/Taq_polymerase). It is a very important ingredient of the polymerase chain reaction. The polymerase chain reaction (PCR) is a technique widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, hereditary studies, paternity testing, and many other applications. The name, polymerase chain reaction, comes from the DNA polymerase used to amplify (replicate many times) a piece of DNA by in vitro enzymatic replication. The polymerase chain reaction, is a technique by which any piece of DNA can be quickly amplified, or copied many times without using cells. The DNA is incubated in a test tube with a special kind of DNA polymerase, a supply of nucleotides, and short pieces of synthetic single-stranded DNA that serve as primers for DNA synthesis. With automation, PCR can make billions of copies of a targeted segment of DNA in a few hours, significantly faster than the days it takes to clone a piece of DNA by making a recombinant plasmid and letting it replicate within bacteria. Amplifying a tiny DNA sample a billion fold does not produce much DNA, but it can be enough for some purposes, such as DNA fingerprinting for a murder trial.(Source: www.dnalc.org/ddnalc/resources/pcr.html). The polymerase chain reaction is used by a wide spectrum of scientists in an ever-increasing range of scientific disciplines. In microbiology and molecular biology, for example, PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology, to name but a few. In clinical microbiology laboratories PCR is invaluable for the diagnosis of microbial infections and epidemiological studies. PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required; for example, sufficient DNA can be obtained from a droplet of blood or a single hair. Just as impressive as the speed of PCR is its specificity. By their complementarity to sequences bracketing the targeted sequence, the primers determine the DNA sequence that is amplified. PCR can be used, for example, to amplify a specific gene prior to further cloning in cells. The PCR makes the gene by far the most abundant DNA fragment, thus simplifying the later task of finding a clone carrying that gene. In fact, PCR is so specific and powerful that its starting material does not even have to be purified DNA. Only minute amounts of DNA need be present in the starting material, and this DNA can be in a partially degraded state. Note, however, that PCR cannot substitute for gene cloning in cells when large amounts of a gene are desired. Occasional errors during PCR replication impose limits on the number of good copies that can be made by this method.
ReplyDeleteDevised in 1985, PCR has had a major impact on biological research and biotechnology. PCR has been used to amplify DNA from a wide variety of sources: fragments of ancient DNA from a 40,000-year-old frozen woolly mammoth; DNA from tiny amounts of blood, tissue, or semen found at the scenes of violent crimes; DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders; and DNA of viral genes from cells infected with such difficult-to-detect viruses as HIV.
DNA forensics took a big step forward when the Taq polymerase was isolated from the bacterium Thermus aquaticus, and when used with a heated strand of DNA and some free deoxynucleotides , these elements cause the Polymerase Chain Reaction (PCR).
ReplyDeleteThe impacts of PCR are enormous. Quite simply, it makes detective work a lot easier. It can be used to match the skin of a suspect found under a victim’s nails to the suspect’s DNA to confirm a killer, something that wasn’t possible before. It can be used to solve paternity cases in a snap. It also can help us determine our genetic code through a method called the Sanger Method.
Developed in 1975, the Sanger Method became the top way of analyzing the genes, due to its simplicity. It work a little like this: Instead of the nucleotides you use in PCR, dideoxynucleotide triphosphates (ddNTPs) are added in to a PCR machine with the deoxynucleotide triphosphates. Four different machines meant that all four nucleotides could be tested for. DdNTPs cut off a chain, as they do not have another ribose to attach it to, and so in the randomness of PCR, there would be some incredible short chains, some incredibly long chains, and then chains just in the middle. These DNA fragments were put into a gel electrophoresis chamber, and by where the fragments lined up, Sanger could determine the DNA of a certain part of a gene, with less radioactive chemicals than before, and faster than before. Thus, the Sanger method taught us a ton about the DNA of our beings in general- it was one of the chief forces behind the National Genome Project, due to its ability to quickly synthesize genes.
http://en.wikipedia.org/wiki/PCR
http://en.wikipedia.org/wiki/Sanger_method#chain-termination_methods
In the hot springs at Yellowstone National Park, a heat stable bacterium called Thermus aquaticus was found. Although it may seem like a small discovery, it opened the doorway to new discoveries in science that had never been thought possible. The important characteristic is that the bacteria is heat stable. Where most enzymes in cells will denature due to high temperatures, the Taq DNA polymerase is used to temperatures of 75-80C. This allows scientists to use the Taq enzyme in the process known as the polymerase chain reaction (PCR).
ReplyDeleteIn PCR, a segment of DNA is replicated by in vitro enzymatic replication. Scientists use the Taq enzyme in order to perform PCR, due to the high temperatures that must be attained during the process. PCR begins with a small reaction tube that is filled with multiple substances. There is the buffer solution that allows for appropriate conditions for the DNA polymerase. There is also the DNA template, which is the strand of DNA that will be amplified, and also the Deoxynucleoside triphosphates that are the building blocks in creating replicas of the template. Then there is of course the Taq enzyme that allows for the denaturing of the DNA strands. To begin the process the reaction tubes may be placed in a thermal cycler and heated, cooled, and reheated numerous times in order to allow for amplification to occur. The tube is first heated to temperatures as high as 98C for 20-30 seconds. This allows for the hydrogen bonds between the DNA strands to break and causes the double stranded DNA to denature and become single strands. After this the temperature is brought back down to around 50-65C to allow for the DNA strands to anneal. Some of the Deoxynucleoside triphosphates bases are attached to complimentary bases of the template strand and create a new double stranded DNA. Then elongation occurs at a temperature of 72C for the enzyme Taq. This concludes a cycle of PCR and cycles are normally repeated in order to create a vast number of replications.
In the field of science and research the process of PCR is a very valuable tool. In forensics, a small sample of DNA that may be used as evidence can be put through the process of PCR and amplified to give a large sample size in which tests can be taken. This is also possible for samples of ancient DNA from such things as fossils. This DNA sample, no matter how small, can be amplified to allow for more tests and studies to be run and a larger sample size. Disease detection is another use for PCR, amplifying early cases of a disease and being able to detect it from the amplified sample size.
PCR is also used in the Sanger method and gel electrophoresis. In this process a primer is places into four separate reaction tubes. These tubes each contain the four deoxynucleotides, but they also contain each a different dideoxynucleotides of each base. This means that when the template is amplified, there is the chance that instead of a normal deoxynucleotide attaching to the template a dideoxynucleoide may attach instead and cause the termination of sequencing. The sequencing would end because the dideoxynucleotides has a 3’H instead of a 3’OH group. If the template is amplified enough times there would be all different variations of sizes of the DNA, each where certain letters are placed. Then the samples may be placed into a urea gel and undergo the process of gel electrophoresis. In this process there is a positive and negative end of the gel. The DNA fragments are placed at the negative end in small wells. Once the electricity is turned on, the fragments will migrate across the gel. Because of the size variations, the smaller fragments of DNA are able to migrate further along the gel and bands are created showing the multiple lengths of different DNA fragments. This process can be used for multiple applications, such as encoding different DNA fragments and also as paternity tests and testing for DNA matches in criminal cases. PCR has expanded the abilities of science to work with DNA and better understand what each segment and compilations of bases does in the human body.
http://www.pnas.org/content/85/24/9436.abstract
http://taqpolymerase.net/
http://en.wikipedia.org/wiki/Taq_polymerase
http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Procedure
http://www.csun.edu/~hcbio027/biotechnology/lec3/sanger.html
http://www.dnalc.org/ddnalc/resources/electrophoresis.html