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We performed a DNA extraction with the same protocol that we previously followed. We then used this extracted DNA, the PCR primers we ordered, and a PCR program we made which followed typical protocol to run a PCR reaction. PCR requires multiple, ordered cycles of denaturation, annealing and extension, of which different temperatures and time periods are used. The denaturation step melts the DNA, causing the DNA to split into single strands. The annealing step then lowers the temperature enough for the primers we selected to bind to the now single stranded DNA at a specific location in the sequence. The extension step then allows polymerase to add dNTPs to the growing complementary strand, effectively adding complementary strands starting from our primers.  This cycle must be run continually for many cycles to amplify our gene’s DNA. After the PCR reaction we ran a 1% Agarose gel electrophoresis to confirm that it was successful and that our DNA was amplified. This allows us to compare bands of PCR product DNA to molecular weight standards to identify our DNA and PCR primer in the gel. We then cut out the band we had identified as our amplified DNA and purified it. This gave us extremely pure DNA for our gene which we then sent to a lab to be sequenced.

Using RNAi, we can use a mutagen to silence any gene in almost any organism, enabling us to perform reverse genetics to analyze genes. Sodium azide (NaN₃), a chemical commonly used to cause a transition from an AT to a GC was used on our as a mutagen on our mutant B. distachyon seeds. Sodium azide functions in chemical inhibition of DNA repairs, often creating a base substitution. These changes can result in various mutations including silent, missense, nonsense, or frameshift mutations. In the DNA sequence, we looked for high impact mutations in the coding regions, splice sites, and donor sequences. We found a high impact mutation in our gene that we determined was a nonsense mutation. A nonsense mutation is a point mutation in which a single amino acid is introduced that changes the codon to a stop codon. Stop codons include the amino acids UGA, UAG, and UAA. These codons terminate protein synthesis and thus, a mutation for a stop codon can have a huge impact on an organism (Scovell, 2018).

After having created a working map for our genomic DNA, identifying our gene of interest and doing extensive research on its possible functions, we needed to perform several experiments to find out more about our gene. The purpose of this lab was to use a reverse genetics approach to explore our Brachypodium distachyon gene of interest and its function by experimenting with mutant and wild type samples. We effectively worked from the mutant phenotype to explore the gene’s function. In doing so, we took several approaches to examine the phenotype, genomic DNA, phylogenetic relationships, and histology of the samples to identify the gene’s possible functions.

Using RNAi. We are now able to silence any gene in almost any organism, enabling us to perform reverse genetics to analyze our gene. Sodium azide (NaN₃), a chemical commonly used to cause a transition from an AT to a GC was used on our gene. Sodium azide functions in chemical inhibition of DNA repairs, often creating a base substitution. These changes can result in various mutations including silent, missense, nonsense, or frameshift mutations. In the DNA sequence, high impact mutations were targeted in the coding regions, splice sites, and donor sequences.

Introduction

A mutant in its basic definition is a genetic variant that causes a phenotype that differs from what is usual for that species. Often, an approach that is used for identifying mutants is called forward genetics. In forward genetics, a gene function is identified by working from a mutant phenotype. However, in pot-genomic era studies, we often employ what is called “reverse genetics”. In reverse genetics, we start from a plant which is homozygous for a mutant allele of an unknown gene and then analyze that gene. We used our B. distachyon plants which were homozygous for a mutation in our unknown gene, analyzing the phenotype of our plant, to help hypothesize a function for the unknown gene.

Clearly, the use of ADHD medication illegally, to enhance academic performance becomes an arms race of who is, and who isn’t taking the drugs. If you aren’t using the cognitive enhancing medication, you put yourself at a major disadvantage. Using these drugs becomes a moral issue firstly because it gives those who take it an unfair advantage. But to go further, these enhancements are also morally wrong because it would likely lead to more difficult course requirements. Just as in sports with anabolic steroids, the issue becomes one of deciding to take the moral path and suffer statistically, or to use enhancements to be at the top.

This method of enhancement can allow users advanced levels of focus and help them stay alert longer to prolong studying. Clearly this presents a great attraction to college students who are desperate to meet deadlines and receive good grades. But the problem arises of whether it is truly fair for some students to abuse this medication to do better in their classes. The problem reflects a watered-down version of the anabolic steroids in professional sports debate. How can it be fair for “natural” athletes or students to compete with these enhanced individuals? If 30% of students are able to pull off all-nighters thanks to these medications, students who are not “enhanced” are faced with quite the handicap. This becomes especially problematic when considering that if these students who are taking ADHD medication are then able to do better than they would without the drugs, this would likely raise the class average performances. If teachers see that their students on average are doing much better on exams, they may feel the need to make the material more difficult.

Case 2

               Across the country, millions of students spend countless hours studying for what seems like endless assignments and exams. Some of these students decide to turn to ADHD drugs such as Adderall and Vyvanse. These drugs are schedule II substances which are available by prescription only. They enhance focus and can be used to stay awake by raising levels of neurotransmitters such as dopamine, epinephrine, and norepinephrine. Studies have shown that full time college students are twice as likely as students who did not go to college full time to take Adderall without a prescription. These studies have also shown that abuse of these ADHD drugs is much more common at more elite colleges. A second study found that about 30% of college students have used these stimulants non-medically, mostly for help studying (Yanes).

Case 2

               Across the country, millions of students spend countless hours studying for what seems like endless assignments and exams. Some of these students decide to turn to ADHD drugs such as Adderall and Vyvanse. These drugs are schedule II substances which are available by prescription only. They enhance focus and can be used to stay awake by raising levels of neurotransmitters such as dopamine, epinephrine, and norepinephrine. Studies have shown that full time college students are twice as likely as students who did not go to college full time to take Adderall without a prescription. These studies have also shown that abuse of these ADHD drugs is much more common at more elite colleges. A second study found that about 30% of college students have used these stimulants non-medically, mostly for help studying (Yanes).

-        Aliens example: one example provided is of aliens from a faraway galaxy which are strikingly like humans. These aliens are not only interfertile with humans, but they are genotypically similar. However, the authors suggest that these factors alone would not be enough for us to consider the aliens to be a member of our species (657).

Gull example: In this example, the authors discuss the herring gull and black-backed gull. Both species of gull are found in Europe but are genetically distinct and cannot breed. However, the black-backed gulls found in North America are much more similar morphologically and genetically to herring gulls. If we were to then classify the North American black-backed gulls and the herring gulls, by transitive property, the black-backed gulls of Europe would then be of the same species as herring gulls. The authors point out that if species sameness is enough to link the two gulls, but they cannot be linked because they are not interfertile, it presents a counterintuitive dilemma with species classification.