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What is Recombinant DNA Technology?

Biology 343

Instructor: Dr. Rinehart

To understand recombinant DNA technology and genetic modification, we must first understand DNA, or deoxyribonucleic acid. DNA, our genetic material, is a double helix made of nitrogenous bases, held together by hydrogen bonds and a sugar-phosphate backbone. This double helix, with antiparallel strands of genetic information, was first realized through x-ray crystallography because of Rosalind Franklin—who, at the time received little recognition for her achievement. Its structure is precise, consistent in dimension, and so simple yet so complex at the same time. The base pairs, thymine, guanine, cytosine, and adenine combine to form genes, an instruction manual for our bodies. The outside of the double helix is formed by the sugar-phosphate backbone and the inside is held together by hydrogen bonds formed between guanine and cytosine, and adenine and thymine. Genotype is based on which genes we have, it is the genetic influence that determines our phenotype; phenotype is the resulting visible characteristic an individual develops—i.e., blue eyes. There are recessive alleles and dominant alleles and, in general, one must have two copies of the recessive allele (one from mom and one from dad) and no copy of the dominant allele to present the recessive phenotype. While some phenotypical characteristics are determined by dominant vs. recessive alleles, some are not so simple. Traits such as height are a combination of our mother, father, and hormones that prompt our bodies to grow. According to the PBS documentary, Cracking Your Genetic Code, there are over 180 genes involved in height. An even more complex concept is the idea that genes can be turned on or off by environment—they can be expressed or not. What I have discussed thus far is still an oversimplification of a highly complicated, and not fully understood, biological system.

Our DNA lives in a protective double-membrane structure called the nuclear envelope. The outer membrane is continuous with the endoplasmic reticulum. Many of our cells replicate—like the digestive track cells and even the skin. Each cell has DNA in the nucleus (each cell that has a nucleus that is), but rather than risk losing or harming this precious DNA, we transcribe the genetic code, making an RNA template that we then translate into the proteins that are essential to many of our bodily functions. To be more specific, there is mRNA and rRNA. M-RNA, or messenger RNA, is a long nucleotide sequence that resembles half of the DNA double-helix; It carries the coded information to the cytoplasm of the cell where protein synthesis can occur. The importance in accuracy of these processes becomes abundantly clear when a mistake is made and a gene is mutated, deleted, or in one way or another rendered mute. For instance, cystic fibrosis (the most common deadly genetic disorder of North America) is the result of an issue as simple as a mis-shaped protein that cannot exit the endoplasmic reticulum as it should, so it is destroyed. This protein is responsible for the membrane channel that allows Cl- enter and exit the cellular membrane. Without this properly working gene our bodies create thick, viscous, mucus that covers our lungs and provides a home for bacteria. Because this thick mucus cannot be easily coughed up, airborne bacteria breeds causing predisposition to respiratory infection. This one gene, CFTR, can create havoc if it does not work properly. The respiratory system is not the only system that is affected in cystic fibrosis, the pancreas, for instance, can become clogged which affects our ability to properly digest food. If we could just replace this gene, would that be an acceptable use of genetic technology? Does it make a difference if we modify this gene in a way that can be passed down to future generations, as opposed to modifying it in a way that affects only the person that is suffering from the lethal disease? If it is ok to do this with CF, what about other diseases? How do we determine the difference between a trait and a disease or disorder? Is deafness a trait? While many people who can hear, see deafness as a hindrance, many people who were born deaf see it as a trait that they would not change. Scientific discovery is quickly advancing, making genetic modification an important topic for discussion.

 While type 1 diabetes can be an autoimmune disease, it can also be a genetic disorder, characterized by the inability to create insulin. Individuals with type I diabetes once had an expiration date, generally dying from diabetic ketoacidosis. Now, scientists have harnessed to power to create insulin through recombinant DNA technology. This can hardly be seen as a negative consequence of recombinant DNA technology, but what are some of the other uses?

First, I’ll begin by explaining what is meant by “recombinant DNA”. Recombinant DNA is as exactly as it sounds, the cutting and recombining of DNA. More often, you will hear the replication of DNA referred to simply as cloning. In order to do this, we must find a way to cut and replicate this DNA or RNA. RNA is nearly the same as DNA, however, it differs in that the nitrogenous base pairs consist of guanine, cytosine, uracil, and adenine. Uracil is similar to thymine in structure, so as thymine pairs with adenine in DNA, uracil pairs with adenine in RNA. Sequences of DNA can be cut by molecular scissors referred to as restriction enzymes, or restriction endonucleases. These restriction enzymes recognize a sequence of base pairs and cut at the desired location. There is a specific type of offset cut that can be accomplished that will leave sticky ends, making it particularly easy to stick chosen sequences together. The use of DNA ligase and ATP can be used to repair the sugar-phosphate backbone to further secure this molecular structure. Beyond restriction enzymes, we also need a vehicle, or carrier molecule, to put the sequence of interest in for propagation. This can be accomplished with the use of a vector. There are various vectors available that include, plasmids, viral vectors, and artificial chromosomes. Plasmids are not found in mammals, but they have proven themselves quite convenient for many recombinant DNA activities. Plasmids are found in bacteria and once reintroduced to their host, rapidly multiply. We have to consider the fact that these molecules are not visible, so scientists have to get creative in order to discover whether or not the plasmid has taken up the sequence of interest. A good vector will make the scientists job easier, it will have an origin of replication, selectable/screenable markers, and many unique restriction enzyme sites. An MCS, or Multiple Cloning Site, is a region of the DNA that contains many restriction-sites that can be cut with different restriction enzymes. The selectable markers either eliminate the nonrecombinant DNA before it colonizes, or they give scientists a visual marker. Antibiotic resistance is a common tool used to kill the nonrecombinant plasmids. If we incorporate a gene for antibiotic resistance, we can then add an antibiotic to the agar plate and only the plasmids with resistance to that specific antibiotic will survive. The simultaneous use of multiple types of antibiotics and antibiotic resistant genes can further help scientists deduce which combinations were successful. PUC18 is a commonly used plasmid that uses antibiotic resistance and color to help identify successful combinations. The lac-Z gene encodes beta-galactosidase. When exposed to X-gal, cells that express the enzyme will convert the x-gal and turn the colony blue. If scientists have interrupted the lac-Z gene and successfully inserted a sequence, the x-gal will not be converted and will not turn blue.

Now that we have a better grasp of how to create recombinant DNA and replicate a sequence of interest, what can we do with it? It took many years to accomplish, but we have now successfully mapped the human genome. This does not mean that we know what every gene does; to the contrary, we still have many questions about the genetic code. Gene hunters work tirelessly, searching for the genes responsible for the worst of the genetic disorders. But we have come a long way, we now recognize genes involved in many genetic disorders and because of this we are able to diagnose and provide targeted therapies for many genetic illnesses. For example, if there is a history of breast cancer in her family, a woman may choose to go and get tested to see if she carries the alleles commonly associated with breast cancer. On the other hand, many women choose against doing so, as they feel that it changes the way they will live their lives. These decisions are very personal and must be made subjectively. There are also genetic disorders for which there are no hopeful treatment options, and many people are uncomfortable knowing whether or not they will experience these types of disorders. Huntington’s disease is one of those illnesses, it has an identifiable gene, but no cure.  

Getting one’s DNA sequenced or genotyped also means that there is now a record of that DNA that can be used in potentially harmful ways (the terms sequencing and genotyping are used interchangeably but they are different). Some have expressed a concern that insurance companies may deny health insurance to potential clients if damning genetic information were made privy to them. It has even been suggested that genetic information may one day be used politically.

One interesting way that this information has been used, is in forensics. While murderers don’t often offer up their genetic material, one of their family members might do so unintentionally. Genetic material is known as “family material”, not only because it is passed down from generation to generation, but because we can see that two people are related, and how closely, just by looking at their DNA. DNA is commonly used to connect people to crimes. What about using a family member to track down a criminal? The infamous Golden State Killer, a serial killer/serial rapist, was caught in just such a manner. Police had DNA from the many crime scenes, but this DNA did them no good without a person to tie the DNA to. Finally, after years of inactivity, the DNA was checked against a genealogy website. People offer their genetic material to these websites in order to learn about their heritage and find long-lost relatives; it just so happens that a relative of the Golden State Killer had used one of these websites and led investigators to him.

While DNA can be used to find relatives, it is also possible to send in DNA for genotyping. Companies such as 23andME use this type of technology to give people answers about genetic risk factors. One issue is that there are many factors that influence the way our genes interact so there are still many questions left open to interpretation. James Watson was one of the first people in the world to have his genome sequenced, and he reportedly indicated that he did not want to know anything about one gene in particular: The APOE gene. APOE codes for a protein, apolipoprotein E. One of the alleles of this gene is known as APOE4, and it is a variant associated with increased risk of late onset Alzheimer’s. This does not mean that if you have APOE4 that you will end up with late onset Alzheimer’s, it has simply been associated with high-risk.

Can we use this newfound knowledge of the human genome to treat people with genetic illnesses? Above, I mentioned cystic fibrosis and the lethal respiratory issues it causes. While 1800 different mutations of the CFTR gene, on chromosome 7, can cause cystic fibrosis, individuals missing as few as 3 letters from the gene are affected—just 3 nitrogenous bases. In 1989, a researcher found the primary variation responsible this debilitating disease. Kalydeco, is a drug that was developed to help improve functioning of the malformed protein of patients with this mutation. Understanding which variant of a mutated gene an individual has, opens up the possibility for these individuals to receive targeted therapies.

What else can we do with recombinant DNA technology? Food is another area of interest. Not just plants, but livestock as well. Plants have been modified for such purposes as to create insect resistance or to make them heartier and more nutritious. Traditionally, plants were modified through selective breeding and crossbreeding, but with the current technology scientists are able to alter plants genetically more easily than ever. The same can be said for livestock. It has been commonplace to breed livestock based upon desired traits, but it is only recently that scientists have harnessed the power of DNA and gained the ability to edit the genetic material of animals. Some of the products of this technology are hornless cattle, goats that carry the human protein lysozyme in their milk, and pigs that have been modified to have resistance to porcine respiratory syndrome. These genetic modifications aim to improve quality, productivity, introduce disease resistance, create environmental sustainability, save animals from painful processes such has horn removal, and save millions of dollars in the process.

I have scarcely mentioned anything negative associated with these technologies, yet many people are skeptical of technology that modifies genetic material, germline and somatic alike. While some are skeptical of the reasoning behind genetic modification, others are scared of the results or claim that scientists are “playing god”. There is a fear that people will start creating designer babies, choosing the most sought-after characteristics. Or worse, they may choose to abort healthy babies based on what they have learned via genetic testing (i.e., aborting a baby because it is deaf). Is this technology inherently dangerous? If not, then what are people so afraid of? Many of the fears are unfounded and based upon misconceptions, misinformation, or simple lack of understanding. I was browsing the internet at one point and saw a post from a lady that said she lost weight simply by eliminating all GMOs from her diet. Some of the information out there is wrong, some is incomplete, and some is difficult to understand. Are there any real concerns when it comes to recombinant DNA technology? The short answer—yes. The COVID mRNA vaccine may be lifesaving, but there are also risks such as: introduction of unknown allergens, cross contamination and migration of DNA among organisms; beyond that, there is also the fear of creating a superbug. This does not mean that recombinant DNA technology is bad and must be stopped, this just means that it needs to be regulated realistically. I’m sure there are people that would pay to create the perfect designer baby if given the option, but that is not an option—as mentioned above, there is complex interplay amongst many different genes, so it is not as simple as just asking a scientist to create a tall, intelligent human. Moreover, even if it was possible to determine all of the characteristics of a baby, regulations could be put in place to stop such things. Misuse of this technology and unintended or unforeseen consequences are fears that are not unfounded, but they are also manageable. We must always ask ourselves if the benefits outweigh the risks and be sure that we are not using this technology to change traits rather than save lives. These types of regulations are being debated as technology steadily advances. This is an important conversation; an open mind and objective outlook are necessary.

Resources:

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.

Marieb, Elaine Nicpon, and Katja Hoehn. Human Anatomy & Physiology. Pearson Education., 2019

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/.

Use of DNA Analysis in Identification, Health, & Human Origins, www.dnai.org/d/index.html.

“Cracking Your Genetic Code .” Cracking Your Genetic Code, PBS. 

“Stem Cell Revolutions . “ , PBS.


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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