Corinne Schuele
Professor Macosko
FYS: Molecular Machines
Nucleotides
February 13, 2008
Taq DNA Polymerase
Polymerase Chain Reaction was developed by Kary Mullis in 1983. Kary Mullis is not the typical scientist accrediting the drug LSD for his discovery. PCR functions as a way to amplify short sequences of DNA usually coding for a specific gene. Polymerase Chain Reaction is like a super fast and efficient copy machine. This genetic copier can create multiple duplicates of the template strand in a short amount of time. According to http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html, the process only takes a few hours to make millions of copies of DNA. It is most widely known for its role in DNA testing for forensic scientists. PCR was famously used to amplify the small amounts of DNA evidence found on the bloody glove in the O.J. Simpson case in 1994. PCR was also used in the mapping of the Human Genome Project. The main molecular machine used in a Polymerase Chain Reaction is DNA polymerase which is an enzyme that aids in the replication.
The solution for a Polymerase Chain Reaction contains a large supply of the four nucleotides, large number of the primer sequence, and DNA polymerase. Using an original piece of DNA as a template, DNA polymerase creates a new DNA strand. DNA polymerase is essentially an enzyme that binds to the template strand of DNA and providing an environment that is suitable for the nucleotides to bind to the template strand. DNA polymerase encapsulates the template strand as if it were a hand grasping it. It then catalyzes the reaction of the nucleotides bonding with the template strand. In Polymerase Chain Reactions, a heat stable polymerase, which was isolated from bacteria living in
The first step in a Polymerase Chain Reaction is to heat the DNA between 94 – 96 degrees Celsius to unravel the double helix and separate the strands. Heating the DNA to this level dissembles the DNA backbone and denatures the hydrogen bonds between the paired bases. Helicases in nature divide the strands. They are the enzyme that essentially “unzips” the DNA strand. Yet, helicases are not a part of Polymerase Chain Reactions. The heating process replaces the need for this enzyme and uses thermal energy to split the DNA strand. Then, short primer strands are annealed to the single stranded DNA templates. DNA polymerases cannot copy DNA without an oligonucleotide primer coding for the beginning of a replication site. Oligonucleotide primers are just short sequences of certain nucleotides that code for the beginning of DNA synthesis for DNA polymerase. As seen on the previous page on the bottom, this is the stage in which the primers can attach themselves to the correct corresponding sites on the DNA template strands. The temperature is lowered so that the primer can create stable hydrogen bonds with the complementary nucleotides. The DNA polymerase then attaches itself to the DNA template and proceeds to copy the DNA.
The temperature is then raised again to create an ideal environment for the enzyme to catalyze the binding of nucleotides to form a complementary DNA strand. If the process was started with one strand of DNA, by this step there would be two new DNA strands.
The temperature is then lowered a final time. The process is then repeated several times to create multiple new strands of the same DNA. Once millions of strands have been created, there is enough mass to run the results of the Polymerase Chain Reaction on electrophoresis gel. PCR allows the small fragment of DNA, usually representing a certain gene, to have enough mass to move through the electrophoresis gel. DNA is slightly, negatively charged. DNA fragments are separated by length when they move slowly through the agarose gel which sits in a buffer so that a slight positive charge can be applied. The agarose gel is finely porous material. The fragments of DNA move along where the shorter move faster because they fit through the holes better. There is always a DNA ladder added where the fragments are of known amount of base pairs. It is used to approximate the size of the unknown DNA. The figure above from http://commons.wikimedia.org/wiki/Image:DNA-Leiter.jpg is the result of a electrophoresis gel. The furthest column to the left is the DNA ladder with known amount of base pairs.
Thermostable Taq polymerase as seen in the picture above is most commonly used for polymerase chain reactions. The DNA is held in the center along with the nucleotides. The reaction is then catalyzed lowering the activation energy required to bind the nucleotides to the template strand of DNA. Taq polymerase is extracted from the bacteria thermus aquaticus. Because if its thermophilic nature, it operates best in high temperatures. It can withstand the denaturation step of PCR in which the solution is heated to extremely high temperatures. With the use of Taq polymerase, new DNA polymerase does not have to be added each time one wants to copy DNA. Kary Mullis discovered the important use of this enzyme and therefore was accredited the discovery of Polymerase Chain Reactions although many others were involved in the discovery.
Taq polymerase lacks a 3’ – 5’ exonuclease. One component of most DNA polymerases is that they possess the ability to correct their mistakes made during the copying of DNA. Molecular machines called exonuclease rectify these mistakes by moving up and down the template strand finding the mutations and eliminating them. Because Taq polymerase lacks this element, it has a high error rate approximately 1 x 10-4 to 2 x 10-5 errors per base pair. This could potentially cause damaged results when ran on an electrophoresis gel.
DNA polymerases vary very little between different species. They all essentially have the same function as a molecular machine helping in the replication of DNA. Taq polymerase is derived from a bacterium, yet functions the same by copying human or any other specie’s DNA. This evidence may be used to show that all living beings have one common ancestor. Because DNA polymerase is a necessary and simple enzyme used in every living organism that requires DNA, it shows that a simple common ancestor possessing the same qualities may have existed. When a complex human genome can be replicated just the same with a bacteria’s DNA polymerase as the human’s DNA polymerase, it suggests the possibility of a simple organism that supplied the basis for all organisms.
DNA polymerase is an essential molecular machine for the genetic code. It provides an ideal environment so nucleotides can bond to DNA fast making copying of DNA a simple and quick process. It also can correct its mistakes which could be detrimental to our health. Without it, our cells would not be able to copy their DNA in an efficient manner.
. "http://en.wikipedia.org/wiki/."
. "http://gaolab.chem.uh.edu/res6.htm."
. "http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html."
. "http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html."
. "http://www.genome.gov/10000207."
. "http://www.pcrstation.com/pcr-primer/."
. "http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.html."
. "http://commons.wikimedia.org/wiki/Image:DNA-Leiter.jpg"
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