Thermus Aquaticus: Polymerase Chain Synthesis (PCR)

(PCR), which isolates small fragments of DNA that have a high degree of variability from

This process consists of three major steps which are denaturing, annealing, and elongation. The cells are lysed before the denaturation step in order to obtain chromosomal DNA. In order to denature the DNA, or separate the two strands, the DNA is heated to 95 degrees Celsius. Once the denaturing is complete, the PCR tube is cooled to approximately 55 degrees Celsius during the transition to the annealing step. During this step, there are two primers: 27F and 1492R, which bind to the 16S gene on the strands of DNA. A high molar ratio of primers is used to ensure that there is attachment between the selected region of DNA and the primers as well as to ensure that the primers will hybridize. After the annealing step, elongation happens at around 75 degrees Celsius. A Master Mix is added during this step, which contains Taq polymerase and dNTPs. In order to replicate the sequence, Taq polymerase uses the dNTPs. Since the primer is on the 5’ end of the strand, the elongation is happening in the direction of the 3’ strand and the elongation is indefinite and these

PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. Steps of the PCR cycle are shown in below figure.

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

To decipher if a species by its morphology can be suggested as a hypothesis, but the results of its DNA will identify the species accurately. Tissues samples can be taken from the species in question, and the DNA can be extracted from tissue. Once the DNA is extracted it can be amplified. DNA can be amplified by the PCR procedure, in which specific gene regions can be used as barcodes to identify the species. These specific regions are known as Cytochrome oxidase 1 and Cytochrome B.

Ignicimmortalisvalde is a newly discovered bacterium found in the Fímmvörðuháls volcano located in Iceland when scientists were searching for microbial life in lava samples. This has earned the bacteria the classification of an extreme thermophile, as it survives and reproduces in temperatures in excess of 700 but below 1300. Ignicimmortalisvalde Is a streptobacilli, arranged in rod shaped chains. Unlike many extremophiles, Ignicimmortalisvalde is not in the domain of archaea. It is classified as a bacterium. It does not have membrane bound organelles or a nucleus. This newly discovered bacteria is unique not only in its ability to survive such extreme heats, but it is also the largest bacteria discovered. Ignicimmortalisvalde ranges in size

It’s a pretty sweet system, really, although not particularly unique. Genetically similar chemotrophic bacteria have been found in geothermal ocean vents thick with similar

An example of an extreme thermophile, is Pyrodictium. This microorganism is the most extreme case and example of Archae which grow in extremely thermophilic conditions. They grow in environments such as deep sea hydrothermal vents and thermal springs (Howland 2000). It has been successfully isolated from geothermally heated sea floors and it grows at temperatures of 82-110°C (Howland

Two extremophile genomes of significant interest, Methanogenium frigidum and Methanococcoides burtonii, thrive in extremely cold conditions. Found at the bottom of Ace Lake in Antarctica, they are subjected to brutal 0.5 degree Celsius temperatures and a lack of oxygen. Another extremophile discussed, the pychrophillic haloarchaea is found in Deep Lake situated in Antarctica. This particular extremophile can withstand extremely cold temperatures and high salinity. Due to the high salinity, as well as the below-freezing temperatures of these lakes, the organisms that occupy them would therefore have had to develop adaptations in order to survive such extreme conditions.

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.

This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and

Due to Taq polymerase being able to tolerate much higher temperatures then that of E.Coli, it is now widely used. Adaptations of DNA polymerase has advanced to AmpliTaq Gold polymerase, this enzyme is inactive before it is added to the PCR. Once it is added to the PCR is only becomes active at 95 °C, this reduces unwanted by- products of PCR that can happen when using Taq polymerase (Goodwin et

Geothermal hot springs are naturally occurring geological phenomena widespread on Earth’s surface (Kormas et al., 2009). Environmental conditions for each geothermal hot spring can vary widely, even between neighboring sites (Oliver et al., 2011). Differences can be observed, for example, in chemical composition of spring water, ranges in temperature and pH, and levels of salinity and other mineral deposits (Jones and Renaut, 2013). According to Stan-Lotter et al. (eds.), “They [geothermal hot springs] can be regarded as islands, ecologically separated by large distances and physiochemical dispersal barriers” (p. 37). A combination of these factors help make geothermal hot springs unique as microbial habitats. However, one overarching similarity among geothermal hot springs appears to be the pattern of organisms that tend to inhabit these sites: thermophilic microbes. Thermophilic microbes thrive at fairly high temperatures, with optimal growth ranging between 55 and 80 °C (Lopez et al., 2013). While several studies have recognized thermophilic microbes belonging to the Bacteria domain, and their respective viruses (Kormas et al., 2009; Grogan, 2013; Bhatia et al., 2015), much of the reviewed literature on hot-spring microbiota have focused particularly on the Archaea domain, and their respective viruses, as they tend to dominate extreme thermal environments (Mochizuki et al., 2010; Pina et al., 2011; Bhatia et al., 2015; Snyder et al., 2015).