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Clostridium Perfringens: The Agricultural Significance and Hazards Posed

Clostridium perfringens: The Agricultural Significance and Hazards Posed when Virulence and Resistance Combine
Clostridium perfringens is among the most ubiquitous pathogens known. C. perfringens is considerably hardy and found in soil, food, water, fecal material, and the intestines of both humans and animals (Uzal et al. 2014). This widespread bacterium is characterized by being a spore-forming anaerobe, which is both Gram positive, and rod-shaped (Kiu and Hall 2018). The importance of this pathogen lies within both its ability to produce a variety of deadly toxins, and the growing concern of antibiotic resistance throughout the globe. C. perfringens is most commonly mentioned for its causation of toxin-dependent infections, such as clostridial myonecrosis, otherwise known as traumatic gas gangrene (Li et al. 2016). However, the complications and breadth of damage that this bacterium causes are not confined to a single method of infection. This highly virulent pathogen has roughly 20 degradative toxins at its disposal, some of which cause more harm than others (Kiu et al. 2017). C. perfringens can infect a multitude of hosts, and each case will vary dependent on the organism, route of infection, and the toxins which are released. While the issues of direct contact with this bacterium are severe, the agricultural impact that is posed by this pathogen threatens nearly every level of food production. From farm fields, to storage, and especially food handling and distribution, C. perfringens cells and toxins are very real threats to quality and safety control aspects of global markets (Hall and Angelotti 1965). These hazards are further exacerbated by the potential for antibiotic resistance to prevent proper treatment of infections. The overuse of antibiotic growth promoters in animal feed for cattle and poultry, among other animals, has led to their antibiotic properties to become ineffective against a variety of pathogens, such as C. perfringens (Gaucher et al. 2017). The ability for bacteria to engage in horizontal gene transfer (HGT) has been extensively studied yet seemingly overlooked and understated in the agricultural business. Keeping farm animals healthy and free of pathogens are among the primary reasons for ample use of AGPs. However, this comes at the risk of stronger, more complex, versions of bacteria to arise and endanger food production (Lacey et al. 2017). Like many bacterial pathogens, C. perfringens ability to alter its outer protein structure when overexposed to certain antibiotics allows for dangerous adaptations in which new antibiotics must be synthesized; which is very costly and not in the interest of many pharmaceutical companies (Uzal et al. 2014). C. perfringens, along with its many toxins and growing resistance to available treatments, is a threat to modern agriculture which must be closely monitored and researched in efforts to prevent outbreaks of resistant strains. It is the responsibility of governments, food producers, and regulatory organizations to understand the potential risks of overusing antibiotics. The agricultural battle to prevent contamination and the spread of C. perfringens begins with properly understanding the arsenal of toxins within both its genome and conjugative plasmids.
C. perfringens toxins are separated into different groups based on the diseases they are associated with, the extent of damage they cause, and which toxin or combinations of toxins are normally employed; the groups are identified as A-E. These categories are used when identifying the four major types of bacterial toxins: alpha, beta, epsilon, and iota (Grenda et al. 2017). In addition to these, there are two other clinically significant types named beta-2 and enterotoxin. These major types are accompanied by 12 minor toxins, which are not attributed to play a major role in disease, but nevertheless are harmful to both humans and animals (Fohler et al. 2017). Each toxin is uniquely identified by the gene it carries, allowing for proper genetic and molecular research into the inner workings of toxin production and pathogenicity. Arguably, the most agriculturally prevalent is alpha toxin. Alpha toxin is the major cause of symptoms and damage associated with histotoxic infections such as gas gangrene (Fohler et al. 2017). Furthermore, the gene which encodes to produce alpha toxin, cpa, is highly-conserved and found in all strains of C. perfringens (Li et al. 2016). In circumstances where alpha toxin is combined with a subtype of toxin, named NetB, avian necrotic enteritis in poultry can occur, which is quickly spread and is both a safety and economic burden (Li et al. 2016). Lesser attributed, yet still important, is the ability for alpha toxin to cause human food poisoning along other gastrointestinal diseases. Beta, epsilon, and iota toxins all have significant roles in agriculture as well.
Encoded by the cpb gene, beta toxin is attributed to the cause of necrotic enteritis in a variety of species. In instances where this toxin is released by C. perfringens near cattle, lamb, and similar species, enterotoxaemia and dysentery can plague whole farms (Fohler et al. 2017). It is vital for the survival of many farm animals that beta toxin be prevented from contaminating animal food; and the spreading of the C. perfringens infections must be identified and controlled in a timely manner. Epsilon toxin, which is encoded by the gene etx, affects sheep and goats. This toxin is best known for its contribution in the cause of enterotoxaemia, more so in the beforementioned animals, but can occur in cattle (Li et al. 2016). Epsilon toxin belongs to groups D and B of the major toxin categories and is not as commonly found in field isolates as the other types of C. perfringens toxins. The last major toxin, iota, is encoded by the iap and ibp genes, and is found only in group E. Iota toxin has an agricultural impact on cattle, sheep, and rabbit (Li et al. 2016). Although not entirely known, it is suspected that this toxin is also a major cause of enterotoxaemia in many farm animals. Along with their many similarities, these toxins are unique in their combinations of a variety of toxin-encoding genes, virulence, and prevalence in the environment. The consequences of animal feed and livestock contamination are the driving force to eliminate C. perfringens vegetative cells and spores from destroying the hard work of livestock producers. In addition to these major toxins, C. perfringens has very significant beta-2 toxin and enterotoxin at its disposal. Beta-2 toxin is recognized for inducing necrotic enteritis in young pigs, human associated non-foodborne diarrheal illnesses, and multiple gut diseases (Kiu and Hall 2018). This potent toxin has various impacts in agriculture and has developing research into its role in human infections. The most important toxin, enterotoxin, is encoded by the cpe gene. Enterotoxin is produced by C. perfringens and is responsible for a variety of human gastrointestinal diseases (Kaneko et al. 2011). Fortunately, enterotoxin is the least prevalent of the toxins produced by C. perfringens. In many outbreaks, enterotoxin is responsible for only 5% of the discovered bacterium population (Kaneko et al. 2011). The significance of this toxin cannot be understated. Enterotoxin is capable of binding to human cells and inducing apoptosis (Kiu and Hall 2018). However, the toxins mentioned here are not only an issue in cattle, poultry, and other animal farms; they pose threats to vegetable producers, storage and transportation, and the handling and distribution portions of agriculture.
While C. perfringens contamination has the potential to begin at an animal breeding facility, it has just as much of an impact and opportunity to do so in the environment. Both vegetative cells and spores can be found on fresh produce, in fecal material, and in various food particles that may not be hospitable hosts; yet these may serve as transmission vectors onto machinery and handler gloves (Bryan and Kilpatrick 1971). Once contamination of a machine, blade, tool, or handler has occurred, the rapid spread of toxin containing cells can be dispersed into such large amounts of food that a catastrophic amount of people can become infected before isolation measures are taken. Ultimately, the introduction of C. perfringens into already cooked foods is of greatest importance (Talukdar et al. 2017). There are a few heat-resistant strains of this pathogen which pose heightened concern among food distributors. Proper cooking may get rid of most C. perfringens cells, however strains which are resistant to normal temperatures of (60° C) may become heat-shocked and induced into rapid germination (Hall and Angelotti 1965). For these reasons, precautions along all areas of food production, from growth and breeding on farms or facilities, to finalizing a customer’s dish, proper sanitation and safety regulations must be followed. Once C. perfringens has been detected, there are methods to determine which strains and toxins are being produced; this is vital to selectively treat the situation. Measures to identify which toxins are infecting animals include the use of the polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (Lacey et al. 2017).
Different methods of PCR, such as nested, real-time, and loop-mediated isothermal amplification (LAMP) PCR, exist to quantify results under various temperatures and bacterial concentration conditions. The goal of these molecular assays is to detect and amplify bacterial DNA for reliable identification of the cause of a pathogenic surge (Kaneko et al. 2011). It is often difficult to determine the source of a C. perfringens outbreak. The methods used are categorized as molecular source tracking, which seek to determine where a toxin-producing strain of a pathogen originated; because C. perfringens is so widespread within the environment, proper determination of a single source of infection requires detailed information on the location where the first patients were infected (Kaneko et al. 2011). Treatment for an outbreak cannot begin until the pathogen responsible is correctly identified. Based on C. perfringens virulent strains and toxins, there is no time to be wasted when a threat has occurred; whether it be on a farm, or in the population, direct action must be taken immediately. Molecular assays, enrichment steps, and gel electrophoresis are useful for the amplification and identification of certain C. perfringens positive foods and isolates when low numbers of bacteria are found (Kaneko et al. 2011). These methods are useful during foodborne outbreaks in which a population has been infected and the pathogen has been linked to a source. Enrichment or induced multiplication of the pathogen is often needed before performing gel electrophoresis, to prevent the occurrence of false-negative results (Lacey et al. 2017). While identification and treatment during outbreaks is very necessary, preventing infection in the first place causes much less of an economic and logistical burden.
Prevention of pathogenic diseases is a very organized and delicate field of regulations, restrictions, and constant observation. C. perfringens and other virulent pathogens must be handled and monitored with extreme care. Certain prevention measures include vaccines, therapeutics, and antibiotic treatments (Kiu and Hall 2018). However, for many pathogens such as C. perfringens, there isn’t necessarily a vaccine that can be taken, and antibiotic treatment has most recently been abused and qualitatively caused more harm than good. Organizations such as the World Health Organization (WHO) respond to and keep a detailed record of previous foodborne outbreaks. It has been estimated that in 2010 there were roughly 3,998,164 foodborne illnesses caused by C. perfringens (Kirk et al. 2015). Preventing the contraction of this pathogen can be as simple as requiring food handlers to properly sterilize equipment after each use, or to wear proper gloves and hairnets. Food contamination begins with improper safety techniques and overlooked risks, with the benefit of speed and mass production, but at the cost of potential outbreaks. However, a variety of chemical agents such as nitrate can be used as preservatives to combat C. perfringens (Talukdar et al. 2017). Research and development are vital in the race against microbial adaptations; finding new methods of inactivating pathogenic spores and preventing vegetative cell growth are just a couple areas of expertise that are constantly evolving. Nitrates, along with organic acids, phosphates, natural antimicrobials and even essential oils have been shown to offer partial resistance to C. perfringens (Talukdar et al. 2017). The most common, yet controversial means of combating microbial pathogens has been the worldwide overuse of antibiotics.
Antimicrobial resistance (AMR) is an emerging threat for various livestock animals and humans. Treatment of many pathogens, including C. perfringens, is becoming increasingly difficult with the emergence of new antimicrobial resistant pathogens (Kiu and Hall 2018). There is a finite amount of antibiotics at the disposal of pharmaceutical and medical professionals. When a pathogen becomes adapted to an antibiotic, it is no longer useful for treatment. Despite this, in the early 2000’s, antibiotic growth promoters were introduced into farm animal feed stock at a staggering amount of 24.6 million pounds for non-therapeutic purposes to prevent contamination (Fair and Tor 2014). This is a pressing issue and is undoubtedly affecting agricultural business both economically and ethically. The preservation and growth of livestock requires a heightened level of control, which one could argue that the use of antibiotics is necessary for preventing mass infection in farm animals; however, this only works if pharmaceutical companies are readily synthesizing new antibiotics which pathogens will have no prior immunity to (Fair and Tor 2014). It is simply not in pharmaceutical companies’ self-interest to produce antibiotics. They are costly to create and are only prescribed for a duration of a couple of weeks. When compared to long-term drugs that treat chronic illnesses, antibiotics are not worth the investment required to pursue (Fair and Tor 2014). AMR has been confirmed in a variety of C. perfringens species which have adapted against tetracycline, gentamycin, and more notably detected the occurrence of mepA, a gene encoding for multi-drug resistance; it is evident that whole genome sequencing techniques will play a vital role in the preparation and development of new antibiotics to treat resistant strains of C. perfringens (Kiu and Hall 2018). Monitoring the everchanging protein structures on pathogen surfaces and staying updated on bacterial genomes are key factors to preventing a future outbreak. The extensive use of antibiotics to keep livestock healthy is not the only cause of growing AMR, doctors have been contributing to its rise for decades.
The over-prescription of antibiotics to treat patient symptoms has played a hefty role in resistant strains of pathogens. The Center for Disease Control (CDC) estimates that 50% of all antibiotics are prescribed unnecessarily, and at a yearly cost of roughly $1.1 billion in the United States, alone (Fair and Tor 2014). Based on these results, changes are occurring in the legal system to prevent catastrophe from AMR pathogens. New programs are arising to address issues of antibiotic resistance. Furthermore, plasmid-mediated conjugative transfer of AMR genes has and will continue to produce more virulent strains of C. perfringens (Gaucher et al. 2017). It is important to note that bacteria have been evolving against natural antibiotics far before any human interference. Bacterial DNA has been isolated from over 30,000 years ago and have been proven to be resistant to natural antibiotic products, and when compared to current strains, bacteria have had much more time to grow in their resistance, than humans have had to synthesize new antibiotics (Fair and Tor 2004). The nature of bacteria, their rapid growth and adaptation, has led to interesting, yet dangerous strains of pathogens to develop. It is a combination of these traits, and the human misuse of antibiotics, which have placed unintentional pressures on bacteria to produce AMR strains, and to ultimately thrive in the absence of antibiotics (Fair and Tor 2004). The most pressing danger lies in C. perfringens ability to transform nonpathogenic strains into virulent, and potentially fatal, necrotic enteritis causing strains. Resistance related genes, virulence factors, multiple enzymes and toxins, have the capability to be included in the accessory genome of C. perfringens (Lacey et al. 2017). This allows for the risk of AMR strain development to arise in the presence of the human misuse of antibiotics; whether by farmers, medical professionals, natural adaptations, or a failure of collective responsibility, C. perfringens will eventually outcompete modern antibiotics.
Food production relies on multiple levels of protection from harmful pathogens, contamination, and quality control. Demand for rises in the production of beef and chicken, along many other foods, has caused an increase in the widespread use of antibiotics (Uzal et al. 2014). Antibiotic resistance is not anything new to this era, and in fact affects many pathogens unrelated to C. perfringens. The attributes which constitute C. perfringens to be an agriculturally significant pathogen include its ubiquitous living conditions, its diverse arsenal of lethal toxins, and its history of economic burden for farmers, medical professionals, and patients alike (Talukdar et al. 2017). The ability for C. perfringens to occupy a variety of living conditions complicates the treatment of foodborne outbreaks. Narrowing the source of an infectious pathogen relies on extensive laboratory testing; this is a challenge with hardy, widespread pathogens. The multiple toxins which C. perfringens has at is disposal further complicates treatment of both animal and human outbreaks. Determining which strain, or if multiple strains, of C. perfringens are involved in a single outbreak is a challenge. However, by identifying the toxins which are released, it is possible to narrow the search and apply known antibiotics or other chemical agents for treatment. The process of inactivating toxins can take years of testing, resources, and capital to pursue (Fair and Tor 2014). Pharmaceutical companies, and researchers alike, have a responsibility to pursue the creation of new antibiotics. Although costly, it is necessary to form new treatments against a variety of pathogens. C. perfringens is just one example that will forever be constantly adapting and forming resistant strains. The agricultural battle to prevent foodborne outbreaks begins with bringing attention to the consequences that will result from a lack of action. Becoming well-educated and raising awareness towards these issues are productive steps, which must be taken, to outcompete C. perfringens ability to contaminate global markets.
Literature Cited
Uzal, Francisco A et al. “Towards an Understanding of the Role of Clostridium Perfringens Toxins in Human and Animal Disease.” Future microbiology 9.3 (2014): 361–377. PMC. Web. 5 Oct. 2018.
Kiu, Raymond, and Lindsay J. Hall. “An Update on the Human and Animal Enteric Pathogen Clostridium Perfringens.” Emerging Microbes

Amplification of DNA by the Polymerase Chain Reaction

Abstract:
In the polymerase chain response, it was consolidated with gel electrophoresis to find the “Alu gene” in DNA using transposons. Polymerase Chain Reaction utilizes certain elements to imitate certain duplicates of a DNA arrangement to help give the gel more “Alu squences” to be resolved simpler. Transposons take into consideration the most widely recognized Alu quality to be perceived using the gel electrophoresis. Each human might possibly contain a chromosome containing the alu quality and some may or may not even have one chromosome that contains the genome. The Polymerase Chain Reaction combined with gel electrophoresis takes into consideration the specific gene that will be identified and in our personal samples to be either homozygous/heterozygous or negative/ positive.
Introduction:
“Polymerase chain response”, also known as PCR, utilizes DNA polymerase to reproduce another strand of DNA from a supplement format. The response needs the accompanying response blend parts: Deoxynucleotides (ddNTPs), Chromosomal DNA, and Taq DNA polymerase; and additionally reverse and forward primers. The thermocycler was required to begin the procedure through the means of tempering, expansion and denaturing. Denaturing utilizes warmth to isolate DNA strands, toughening places the forward and switch preliminaries to set up the expansion stage and the taq DNA polymerase prolongs the strands of the DNA in the expanded stage.
The bits of DNA are “transposons” that can supplant itself in somewhere else in the genome. It is where it can recreate and basically reorder itself into another DNA. “Double-stranded DNA reinsert elsewhere in the genome, i.e., the classic “cut-and-paste” transposons” (Cédric) The accessible and the most available transposons are Alu components which are in the 300bp range. ALu genes are a standout amongst the most copious found in the “human genome at roughly 1 million” (Prayel).
The technique in which DNA strands are assessed by length is obtained by using “Gel Electrophoresis”. Every model is put into a well in the agrose gel where it is subjected to electrical current to ultimately have the DNA strands move towards the anode or positive charge bearing. “Shorter strands can push ahead on account of the manner in which that the gel contains sugar fibers that can obfuscate longer strands and its movements” (Heuer).
As every human is different, some may or may not have the alu gene. Some may even be carriers of the alu gene (Hetrozygous), maybe they have obtained the alu gene from only one of their parents and not the other. Whereas other that are homologous negative, have not received the alu gene from either one of their parents.
Methods:
Everyone in the lab will take an example of their cheek cell and break it down through using the PCR method by utilizing a toothpick and scratching within the sides of the cheek/mouth. Once done, put the finish toothpick that you scratched your cheek cells with. Use a microcentrifuge to obtain .3 ml and mix it with 100 uL of the buffer extraction and whirl it around. Name and top the test tube to exchange to the warm cycler at fifty-five degrees celsius for an hour. Towards the finish of the cycle, for approximately ten minutes, it will be at ninty-five degrees Celsius to inactivate the protein. Recoup and ensure the test tube is legitimately named.
Start to set up the PCR by marking a tube “alu” with your initials and add .2 microliters. Next, add the following: 7.5 uL of distilled water, 1.25 uL of forward primer, 1.25 uL of the reverse primer, 2.5 uL of all the cheek cell genomic DNA, and 12.5 uL of the “Master Mix”. Each table will set up a control .2 microliters of the PCR tube and name it “C” with their initials and instead of adding the genomic cheek cell DNA will be adding 2.5 uL of distilled water. Set the examples in the thermocycler and sit patiently for the teacher to begin it for thirty-five cycles. Record the temperatures and times through one finish cycle 3 times. The denaturing step was set at ninty-five degrees Celsius for an entire hour, and for 60 seconds at sixty-two degrees Celsuis was the annealing step and the seventy degrees Celsius for 60 seconds was also for the extending step.
Proceed forward and obtain the sample, utilizing the class size, make sure the class samples can fit the considerably even # of gels. With the fourteen-well comb put 1.5 percent of agarose gel and give it time to solidify totally. Setting the gels in the electrophoresis chamber closest the dark anodes put 1X TBE and expel the brush. In the most remote left and right segments embed bp ladder of 6 ul. Now, in everyone’s 20 microliter sample, add “four uL” of the dye and from the mixture put “five uL” in the gel. Run the gel for half-an-hour at 120 volts. Once completed, take the gel to the region specified by your TA to put the gel in an UV light box and to see the photo of what is shown.
Results:
The outcomes of this lab demonstrated that various models had particular results in that some were either negative or positive for the gene of the alu. And some could actually be carriers of the gene, and be “heterozygous”. Only four students that were on the left side will be are area of interest for this result section.
As for the markers that were outside wells, “seven and one”, the well four was the control. And well “one and three” showed the alu gene negative homozygous meanwhile for well 4, there was a “alu gene” that was positive homozygous. Also, nothing was showed on the gel for well two and well 3. None of the models were seen to be heterozygous subject to the undeniable social occasions that could be surely watched.

By utilizing the gel electrophoresis, the photo shows the distinctive alu gene pieces. Wells “two through six” were one gathering of four understudies that will be inspected further and wells “nine through twelve” was another gathering of three understudies. Wells “one, seven, eight and thirteen” contained every single marker while “four and ten” were the controls of the examination. Sadly, the personal sample had no alu gene shown on the gel. Wells “two and four” contained groups at the 400bp implying that the examples were negative homozygous for the gene of the alu in the two chromosomes. Well “six” contained another example in which was positive homozygous for the gene of the alu in the two chromosomes.
Discussion:
As for the “Polymerase Chain Reaction”, the results can be better furthered and can be bettered improved through the utilization of the recently created “microfluidic systems”, in which multiple, various reactions can occur simultaneously at once. The Real-time “Polymerase Chain Reaction”, PCR are can be finished in about an hour or less, or, in other words, way faster than the ordinary PCR that has been utilized in this lab. Through the utilization of the “real-time PCR”, it allows for a quicker diagnosis of infectious disease that would otherwise be fatal if it is not figured out in a timely manner (Ahrberg).
The flaws that could have occurred for this lab are infinite and could have occurred at any given time during the duration of the experiment. For instance, the measurements could have been off and as a result, skew the whole outcome of the experiment. Or a simple mistake in the beginning of the lab, not taking enough cheek cells on the toothpick. This may have been because there was a lack of scraping correctly on the sides of the insides of the mouth. As a result, the lack of cells on the toothpick couldn’t be used for the PCR to see the alu genes.
As for the sample that was personal, the photo shows nothing on wells three. Wells two and five had contained samples that showed homogenous negative for the gene of the alu. Meanwhile, for the well six, it showed that the sample was indeed positive homogenous for the gene of the alu. The bands that were faint were not shown because it was impossible to legitimately have the capacity to tell which tests were homozygous or heterozygous for the gene of the alu for those that were faint in color.
Conclusion:
The hypothesis was not ready to be tried in light of the fact that the individual sample was not found or done inaccurately. In any case, utilizing the first sample one, the outcomes were valid that the example would be a homozygous negative for the gene of the alu. The identification of the gene depended entirely upon the individual samples on whether or not each sample had a chromosome for the alu gene. “Polymerase Chain Reaction”, PCR is imperative since it recreates DNA strands using distinctive parts. PCR is precise in separating the DNA strand than reconstructing another supplement.
References:
Feschotte, Cédric, and Ellen J. Pritham. “DNA Transposons and the Evolution of Eukaryotic Genomes.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 2007,
Ahrberg, C D, et al. “Polymerase Chain Reaction in Microfluidic Devices.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 5 Oct. 2016
Alberts, Bruce. Essential Cell Biology. New York, NY: Garland Science Pub, 2017. Textbook.
Prayel, Leslie A. “Functions and Utility of Alu Genes.” Nature News, Nature Publishing Group, 201
Heuer, Tim. “PROTEIN GEL ELECTROPHORESIS.” Oligo-DT Cellulose Columns, 1998,

Questions:
SECTION B: PCR Amplification of Alu Sequences
Q1) Proteins are denatured at high temperatures. So how are we able to perform PCR at these high temperatures using DNA copying enzymes?
We were able to do this because it had a thermostable enzyme called Taq polymerase, in which could not denature at high temperatures.
Q2) What was the annealing temperature that was programmed into our thermocycler? Why was this temperature chosen?
The temperature was sixty two degrees celsius, and the reasoning behind that was because the primers had to start synthesizing DNA at the low temperatures.
Q3) If the sequences of the PCR primers are;
5’-GGA TCT CAG GGT GGG TGG CAA TGC T-3’
5’-GAA AGG CAA GCT ACC AGA AGC CCC AA-3’
Calculate the melting temperature of each, based on the observation that for every A or T add 2oC, and for each C or G add 4oC? Show your calculations.
For the 1st strand we had to do:
– Guanine/Cytosine=15
– Adenine/Thymine=10
– 15×4 10×2=80 C
For the 2nd Strand we had to do:
-Guanine/Cytosine=14
– Adenine/Thymine=12
-14×4 12×2=80 C
Q4) If the extension temperature was eliminated altogether from the thermocycler, what would happened to our Alu PCRs? [Hint: the size of your fragments is important]
The alu PCRs will would become shorter because during the extensions stage, the DNA will become extended so that they don’t shrink. This is to ensure that the final product once done will display the alu genes, showing whether it’s is a either a negative/ positive for the heterozygous or homozygous.
SECTION C: Electrophoresis of Amplified DNA
Comprehension Questions:
Q1. What is a transposon?
A transposon, in essence, is a piece of “Deoxyribonucleic Acid”, DNA that that can replace itself in the DNA sequence.

Q2. How common are transposons in the human genome?
Interesting to note, Transposons are common in the human as there are approximately over a million of them in your very genome.
Q3. How does a transposon “jump” from one site in the genome to another?
They jump from site to site in the genome by a method that is called, “copy and paste”. This helps replicate itself using the mRNA and then cut out DNA pieces and incorporates itself inside.
Q4. What is a polymorphism?
Polymorphism is variation in the DNA’s sequence in which is a common in the population.
Q5. How can PCR be used to determine the Alu genotype of an individual?

PCR is utilized to determine the alu genoype of an individual by the use of gel electrophoresis. By using gel electrophoresis, the alu genotype of a person can be seen in the band length and therefore show whether a sample is positive or negative for the alu gene.
Q6. How is Taq DNA polymerase different from DNA polymerase found in E. coli?
Taq DNA polymerase is different from DNA polymerase found in E. Coli in that , the Taq is a lot quicker in the breakdown of the DNA by using high temperature.
Q7. What is the role of the forward and reverse primers used in PCR?
One of the main roles of the “forward” and “reverse” primers allows the DNA’s target reason to replicate.

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