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Genetics: Recombinant DNA Technology

An advantage of this recombinant DNA technology is the ability to produce these life-saving treatments for worrisome genetic disorders. A diagnosis of type-1 diabetes once came with an expiration date, now individuals with type-1 diabetes can live long, healthy, lives.

I have found a new love! My primary interest is still behavioral neuroscience and psychopharmacology; However, after taking a course called Future of Genetics this term, I’ve become insanely fascinated by the subject. Here’s a homework assignment that covered many of the concepts convered this term =)

Torie Simmons

Biology 343: Future of Genetics

Instructor: Dr. Rinehart

List and describe the basic methods and materials required to cut a sample of human DNA and insert those pieces into a plasmid vector to be propagated in E. coli bacteria. Your answer should include the properties of a good plasmid vector, the enzymes involved and a note about what they do, and a consideration of how you would be sure that the bacteria carried truly recombinant plasmids (selection scheme).

In any good DNA arts & crafts project we need a certain set of tools. First, we need our molecular scissors, also referred to as a restriction enzyme, such as EcoR1. A restriction enzyme, or restriction endonuclease, can make a blunt cut or an offset cut with “sticky ends”. The cell from which the restriction endonuclease was purified will have a protective chemical signal on the target sequences in its own DNA, that modifies the DNA, preventing its own destruction. Foreign DNA that lacks this mechanism can be cut at the specified site. Restriction endonucleases from different bacteria may seek out different target sequences. Varying nuclease enzyme types often recognize nucleotide sequences that are 4-6 base pairs long. Some of these restriction enzymes cut within the target and some simply cut near it; When “sticky ends” are cut, the DNA can be joined easily to other DNA with complementary ends. EcoR1, for instance, leaves two sticky ends, making it easily stick (it forms hydrogen bonds) to a DNA fragment from another source, assuming base pairs are complementary. While the “sticky ends” adhere, there is still a gap in the sugar-phosphate backbone, which must be repaired or ligated with DNA ligase and ATP. Next, we will need a vector. A vector is a carrier molecule that can carry our DNA sequence of interest and allow it to propagate. Different types of vectors include plasmids (not found in mammals), viral vectors, and artificial chromosomes. A good vector must have a few basic characteristics: an origin of replication, selectable markers, and many unique restriction enzyme sites. Many vectors have a region called the MCS, or Multiple Cloning Site, which is a region of the DNA that contains many (generally unique) restriction-sites that can be cut with different restriction enzymes. Bacterial transformation is what we call the process of bacteria taking up a plasmid, because the bacterial cells are being transformed. In order to get E. coli to take up plasmids, calcium chloride solution is added to the mixture before a heat shock is administered; for one reason or another this encourages the process. Selectable markers, and screen-able markers are an important piece of the puzzle, allowing us to visualize successful combinations. Selectable markers essentially kill nonrecombinant cells, and screen-able markers provide a visual marker. One way to recognize the recombinant DNA in a successful transformation, is using antibacterial resistance to our advantage; if we use a plasmid that has a gene for penicillin resistance, the plasmid DNA molecules multiply, and then we spread the bacteria onto an agar plate containing penicillin and incubate it. The penicillin in the agar kills most of the cells and of course the cells that are left must have the penicillin resistant plasmid. If each DNA sequence has different antibiotic resistant properties, we can further eliminate nonrecombinant results. Color has also been used as a helpful identification tool used to screen the DNA, making sure the plasmid has taken up the DNA; the lac-Z gene encodes something referred to as a beta-galactosidase. When exposed to X-gal, cells that express the enzyme will convert the x-gal which ultimately turns it blue. If the colonies turn blue, this may indicate that they did not integrate the DNA insert. PUC18 is a plasmid that was engineered using these tricks, it has a gene for ampicillin resistance, and the lacZ sequence that will be disrupted if the plasmid combines with other DNA.

List and describe three SPECIFIC examples of how genetic engineering has improved productivity in agriculture (including such things as productivity of food animals, not just crops). What are some concerns people have voiced regarding recombinant technology on the farm? What is your opinion on the matter? Is your opinion supported by a logic and analysis of available data, or is it a gut reaction? If your opinion is emotionally based, has what you’ve learned in this class changed how you view the issue?

According to an article titled, Cow. Interrupted, at UC Davis, goats were modified to carry a human protein called lysozyme in their milk. A later realized consequence was that the milk could halt diarrhea. Another improvement ushered in by genetic alteration was polled, or hornless, cattle. Yet another genetic transformation—pigs with resistance to porcine reproductive and respiratory syndrome. Porcine reproductive and respiratory syndrome is an untreatable disease that costs the swine industry millions of dollars every year (Barber, 2019).

Numerous concerns have been voiced, many of which seem to stem from misunderstanding, or fear of unintended consequences. The World Organization for Animal Health voiced concerns about “how an animal is coping with the conditions in which it lives”. According to a PMC article, Genetic Engineering of Animals: Ethical Issues, Including Welfare Concerns (2011), some of the ethical concerns considered include: unanticipated welfare concerns, invasiveness of the required procedures, large numbers of animals required, and how exactly to establish guidelines and limitations as pertains to ethical treatment of animals. One of the problems that scientists have once they have successfully created, what they deem a superior animal, is the costly and timely process of getting the animal approved for market. Why do scientists want to genetically engineer or modify these animals? Is it worth the cost and what is the goal? There are many applications that are meant to improve productivity, quality, disease resistance and environmental sustainability. Some genetic modification simply enhances food quality, as with the goats mentioned above, some are engineered to create disease-resistance in animals, and some are even engineered for such reasons as reduction of pollution.

What is my opinion? I am reluctant to form an opinion—at least a strong opinion that assumes that this technology is either good or bad. Much of the technology has been beneficial and will continue to benefit humanity. I think that there are many advantages associated with these genetic modifications and with proper regulation the world could be better, presuming scientists continue to improve upon their current work; However, I am not a scientist and there are many concepts that I do not fully understand. Unintended consequences can be difficult to foresee and without proper research and education I prefer to avoid forming a hasty opinion, as so many do. I feel that one term of research is enough to open my eyes and elucidate many of the mysteries associated with recombinant DNA and genetic engineering, but I would be foolish to think that I fully understand the consequences. For now, I have found a new interest and plan to follow scientific advancement.

List and describe two examples of how genetic engineering has produced new drugs, vaccines, or improvements of existing drugs or vaccines. For purposes of this question, you may define naturally-occurring hormones, such as insulin and human growth factor, as drugs. What are some advantages and disadvantages to using such methods? Are there any dangers associated with these recombinant drugs?

A popular example of a naturally-occurring hormone being produced for the good human kind is insulin. Not only did Dr. Rinehart mention this in the lectures, but nearly anywhere you search for answers associated with recombinant DNA technology applications related to drugs, vaccines, and hormones, insulin will be mentioned. To make insulin, the gene for human insulin can be identified, cut with a restriction enzyme, inserted into a plasmid vector which is then inserted into bacteria for propagation, and then it can be rapidly multiplied, producing large quantities of the substance that will be used to treat diabetes. Insulin-like growth factor (IGF) is also being developed for IGF deficiency. Rather than discuss IGF, I will focus on another more exciting, yet controversial, vaccine that is of particular relevance currently; the COVID mRNA vaccine is being used to help control the current pandemic. The mRNA is used to create the protein profile associated with COVID so that our bodies will create an immune response to fight COVID.

An advantage of this recombinant DNA technology is the ability to produce these life-saving treatments for worrisome genetic disorders. A diagnosis of type-1 diabetes once came with an expiration date, now individuals with type-1 diabetes can live long, healthy, lives. It would be difficult to argue that this change is not advantageous to mankind, so what are some disadvantages? Well, genetic engineering may cause unintended consequences that are not apparent immediately, such as future allergic reactions. The COVID mRNA vaccine, for instance, may introduce these types of unknown allergens. Cross contamination and migration of DNA among organisms is also a concern. Moreover, many people worry that with the creation of these vaccines, we run the risk of making the virus more virulent, creating a superbug.

You have isolated a gene from human DNA that codes for an important enzyme. Assuming you have cloned the gene, describe how you would use Southern Hybridization to determine if a sample of chimpanzee DNA also contains the gene.

Firstly, I would begin by isolating DNA from the chimpanzee. After isolating the DNA, I would use a restriction endonuclease, such as EcoR1, to cut the DNA into pieces. Next, I would put the DNA fragments into agarose gel using a pipette to drop them into wells that were put into the gel before it solidified. After the DNA has been added to the agarose gel, we can separate the fragments using electrophoresis. Because the DNA is colorless, we have to consider the need for a tracking dye. Once the DNA is tagged with a tracking dye, we can be sure that the DNA actually made it into the agarose gel. This agarose gel is porous and thick so the smaller DNA will move farther in the allotted amount of time, separating the DNA according to size. There is a slight charge present in DNA, so if we use this charge to our advantage, we can move DNA molecules through gel, and they will be separated (based on their size) using electric current. Specifically, the backbone contains a slight negative charge so the molecules will be attracted to the positive charge placed at the other end of the gel. It is worth mentioning, that if we want to separate based on a size difference of only a few base pairs, agarose gel will not suffice, and things become substantially more complicated. For this answer, we will focus on how we would use agarose gel. Ethidium bromide can be used to treat the gel, staining for visibility. The ethidium bromide binds to the DNA and glows when an ultraviolet light is shined on it. According to Dr. Rinehart, there are alternatives to ethidium bromide that are safer for use. Markers are used to compare the DNA fragments by their sizes. The DNA in the gel is denatured and transferred to nitrocellulose paper. Now it is exposed to a hybridization probe that has been tagged.  The radioactive probe contains the nucleotide sequence of interest. After incubation with the probe sequence, nonhybridized radioactivity is washed off the paper and x-ray film is used to detect the position of the probe. As we know, complimentary base-pairing has been a vital tool in learning about DNA; the probe binds to complementary DNA segments and If the chimpanzee DNA binds to the probe, then we can infer that the chimpanzee may also have this gene.

Resources:

*Along with the resources below I also used three course lectures given by Dr. Rinehart and pdf files uploaded to the class.

Bawa, A. S., and K. R. Anilakumar. “Genetically Modified Foods: Safety, Risks and Public Concerns—a Review.” Journal of Food Science and Technology, vol. 50, no. 6, 2012, pp. 1035–1046., doi:10.1007/s13197-012-0899-1.

Damian Garde — STAT and Jonathan Saltzman — Boston Globe Nov. 10, et al. “The Story of MRNA: From a Loose Idea to a Tool That May Help Curb Covid.” STAT, 7 Jan. 2021, http://www.statnews.com/2020/11/10/the-story-of-mrna-how-a-once-dismissed-idea-became-a-leading-technology-in-the-covid-vaccine-race/.

Drlica, Karl. Understanding DNA and Gene Cloning : a Guide for the Curious. John Wiley & Sons, 2004.

Khan, Suliman, et al. “Role of Recombinant DNA Technology to Improve Life.” International Journal of Genomics, vol. 2016, 2016, pp. 1–14., doi:10.1155/2016/2405954.

Ormandy, Elisabeth H, et al. “Genetic Engineering of Animals: Ethical Issues, Including Welfare Concerns.” The Canadian Veterinary Journal = La Revue Veterinaire Canadienne, Canadian Veterinary Medical Association, May 2011, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078015/.

By tbsimmons63

I'm beginning a long and exciting journey. I've been diagnosed with PTSD, Bipolar I, and Anxiety Disorder but I don't feel that any of these things have to define me. I'm planning to document my journey as thoroughly as possible in hopes of helping others find their way!

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