Corinne Schuele
Professor Macosko
Molecular Machines
Genetic Code
Taq Polymerase and PCR
DNA polymerase is the molecular copy machine of the genetic code. It is an essential enzyme which copies and makes new strands of DNA. It grabs a hold of the template DNA strand much like a hand and creates a favorable environment for the new nucleotides to bind. Polymerase catalyze this reaction lowering the activation energy required making the process of copying DNA a fast and efficient one. Without it our cells would be slow to replicate causing detrimental effects on our body.
A DNA polymerase derived from the bacterium thermos aquaticus, called Taq DNA polymerase, is used in most widely in Polymerase Chain Reactions. This bacterium is found in
Although Taq polymerase has many good qualities, it lacks a 3’ to 5’ exonuclease, which travels back and forth on the new strand of DNA looking for errors and then corrects them by cutting out the mistake. This can cause an error rate of 10-4 to 2 x 10-5 errors per base pair which could cause skewed results in a Polymerase Chain Reaction.
There are many types of DNA polymerase in organisms. DNA polymerases are comprised of seven families, yet vary little among each group. They all essentially carry out the same process, the copying of DNA. Thus, all DNA polymerases function in almost exactly the same manner. Hence the reason Taq polymerase derived from a bacterium can copy human DNA.
DNA polymerases can only add nucleotides from the 3’ end of DNA. They can never start a new strand of their own. DNA polymerase cannot replicate unless there is a 3’ OH group attached to the template strand. To add this, a primer of short nucleotide sequence binds to the pre-existing DNA. The primer carries the OH group to the new DNA allowing for the DNA polymerase to recognize and attach to the strand needing replication.
Like most DNA polymerases, Taq polymerases are made up of a finger, thumb, and palm region. The thumb region is made up of COOH-terminus, three a helices, and a two-stranded beta sheet. Hydrophobic reactions and salt bridges create a “hinge” between the thumb and the palm allowing it to move and change conformation shape when the DNA binds to the reaction site. When the DNA binds to the reaction site on the thumb, it closes on the DNA clasping it into place. The palm contains two α helices and a compacted group of beta sheets. The reaction is catalyzed in the finger of the polymerase. The polymerase goes down the DNA in a somewhat pinching motion where the finger and thumb constantly release and bind to the DNA.
http://users.ugent.be/~avierstr/principles/pcrsteps.gif
Polymerase Chain Reactions allow for the copying of short DNA sequences in a small amount of time. This can be used to generate enough evidence for DNA testing for a crime scene. The bloody glove found in the OJ Simpson case was analyzed in this manner. A sample of the blood was amplified using PCR and then ran on an electrophoresis gel next to DNA from OJ Simpson to see if the results matched. PCR can also be used to find mutations or map the human genome.
Like I described in my previous paper, PCR goes through several cycles of heating and cooling where primer is added and Taq polymerase copies the template strand creating many copies of the short sequence. Then after enough DNA is produced, the sample is usually run on an electrophoresis gel. Electrophoresis gel is an agrose gel which is porous allowing for the DNA strands to move through the matrix.
The sample of DNA is added to the well at the end of the gel along with dye so it can be visualized. A DNA ladder is always added in a separate well. The ladder is the farthest row on the left in the picture above. A DNA ladder is comprised of DNA fragments of known number of base pairs. This allows scientists to compare the sample of DNA and be able to sequence it from the DNA ladder. A buffer is added to the electrophoresis gel so that a small voltage can be applied. DNA is slightly negative so the opposite end of the gel is given a positive charge. The DNA fragments migrate to the other side and separate among themselves. The shorter strands will mover further down the gel because it is easier for them to fit through the pores in the gel than the longer strands. The gel can then be analyzed and sequenced by comparing the ladder and the sample.
DNA testing uses this method. If one is trying to solve a crime, the DNA sample found in the crime scene is run on a gel next to the suspect’s DNA. If the bands match, then the suspect’s DNA was the one found in the crime scene. Likewise, this can be used to determine the rightful parent of a child. PCR makes appearances in many soap operas and detective shows because of these applications. PCR can also save lives by detecting deadly tumors. FISH technique is used in which a short segment of DNA is “tagged” by a PCR and a fluorescence gene is added so the gene coding for the cancer can be located.
PCR allows for an inexpensive and fast way to test and analyze DNA samples. It copies short strands of DNA fast and efficiently. Then running the samples on an electrophoresis gel allows for the visualization of results. Taq polymerase or DNA polymerases in general are the molecular machines that power this reaction.
· http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/t7dnapol/molmast.htm
· http://en.wikipedia.org/wiki/DNA_polymerase
· http://en.wikipedia.org/wiki/Gel_electrophoresis
· http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization
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