Thermus Aquaticus: Polymerase Chain Synthesis (PCR)

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

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.

coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.

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).

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.

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

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

The objective of the Box Polymerase Chain Reaction (PCR) experiment was to gain a basic understanding of the Polymerase chain reaction and gel electrophoresis, and then apply that knowledge to identifying the unknown bacteria. We found that our bacteria

NaOH is then applied for cell lysis and the ‘unzipping’ of dsDNA to ssDNA. The ssDNA may then be used to isolate and replicate the PCR product through the use of PCR and site specific primers, using 2 specific primers to isolate both sides and ends of the mtDNA D.loop, multiple runs of PCR are taken to receive multiple copies of the PCR product. The following sequence primers are used to isolate the PCR

Buckinghamshire, UK) were used within each supplied tube. Each bead contains the desired ingredients needed for the successful PCR reaction, stabilizers, bovine serum albumin to increase overall yield, four deoxynucleotides triphosphates (dNPT), adenine, guanine, cytosine, tyramine ATP, approximately 2.5 units of puReTaq DNA polymerase and a reaction buffer. When reconstituted to the final volume of 22µl, the final volume of each dNTP is 176µM in 8.8mM of Tris-HCL which at room temperature has a pH of 9.0. Finally, 44mM of potassium chloride and 0.66mM of magnesium chloride. All PCR was preformed within a fume cupboard (Bassaire 06HB) to minimise the risk of either moisture or genomic

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

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).

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.