Lab 13. DNA Biology and Technology

Note: All your answers to questions must be in Red or other colors (not including blue) for easier grading. Points will be deducted if you do not distinguish your answers. Lab 13. DNA Biology and Technology Objectives:  Extract DNA from a strawberry using household products.  Identify the role of chemicals in the process of extracting DNA.  Observe a large sample of DNA.  Describe the process of DNA gel electrophoresis. Vocabulary:  DNA  Gel electrophoresis  Octoploid  Filtrate  Precipitate  Agarose Gel Introduction: DNA is the instruction manual for living things. By far, the greatest amount of DNA is located in the nucleus of eukaryotic cells and described as a double-helix. The double-stranded genetic blueprint, runs antiparallel, with bases bonding in a complementary fashion, ensuring that with every round of replication or transcription, a parent (or template) strand remains. This semi-conservative replicative strategy ensures the integrity of the code, for the proteins that result from the nucleotide sequence are vital to the cell. Every cell that comprises a living organism contains the complete genetic blueprint of that organism, what enables the specialization of a particular cell in a particular area of the body is control over which genes are expressed and when. As every cell contains DNA, isolating the genetic sequence of an organism from a subset of cells, such as blood, sweat, and skin, to name a few. Once a sample has been obtained, the DNA must be released from the nucleus, which involves the physical disruption of the cell. After the cells have broken open, a salt solution (NaCl) and a detergent solution containing the compound SDS (sodium dodecyl sulfate) are added, to breakdown the cell membrane. Finally, ethanol is added causing the DNA to precipitate (settle out) of the solution, leaving behind all the cellular components that aren't

soluble in alcohol. The resulting DNA can be spooled (wound) on a stirring rod and pulled from the solution at this point. The extraction and purification of DNA are of primary importance to the field of biotechnology and forensics. DNA extraction allows for analysis including the detection of genetic disorders, identification of individuals using DNA fingerprints and studies involving GMOs (genetically modified organisms). The genetic code is universal, which means, traits from one organism can be expressed in other, nonrelated organisms. Genetic engineering is the directed addition of new nucleotide sequences to an organism's genome, which can be employed to increase nutrient content, make animals more resilient, or better adapted for a region. While, often the subject of heated debate regarding safety, restrictions, and ethics, genetically engineered organisms could revolutionize agriculture and healthcare. Modifications could result in bacteria capable of producing insulin, antibiotics, and hormones. Gel Electrophoresis Because nucleic acids are negatively-charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size using this charge and may be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules ; this difference in the rate of migration separates the fragments on the basis of size. There are molecular- weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size. Part 1: Strawberry DNA Extraction/ Do it at home (optional) Strawberries are octoploid , which means they have eight sets of chromosomes. The procedure for extracting DNA from a strawberry is simple, and the results are usually obvious, it is easy to see the white strands of DNA within the pink solution of strawberry juice . In this procedure, you will crush a strawberry and add detergent and salt to break down the cell walls to release the DNA within the nucleus. You will then filter the liquid from this crushed strawberry into a beaker, the substance is

Procedure: F igure 2: This diagram shows the basic method used for extraction of DNA. 1. Add a strawberry (or half) to a Ziploc storage bag. 2. Add 10 ml of the DNA extraction buffer and mash the strawberry and buffer for about one minute. 3. Use a funnel and coffee filters to filter the strawberry juice into a beaker. 4. Transfer the filtrate to a beaker for about 10-15mins, you should then transfer the solution to a test tube and only fill the test tube about half full and avoid transferring any foam. 5. Slowly pour or drip cold alcohol/isopropyl (5mls) over the top of the strawberry mixture. You want a single layer on top of the strawberry mixture. 6. White strands will form in the ethanol layer, use a stirring rod or toothpick to spool the strands. 7. Transfer the spool into a centrifuge tube Discussion: 1. What does DNA from the strawberry look like? The DNA from the strawberry manifested itself as fine and stringy with white bands/clumps. 2. Why is it important for scientists to be able to remove DNA from cells? The ability to extract and analyze DNA is fundamental to various fields

of science and has numerous applications which has transformed our understanding of genetics, enabling advancements in medicine, forensic science, biotechnology, and various other scientific disciplines. 3. What is the role of detergent, ethanol, and salt in the extraction process? The detergent and salt break down the cell walls to release the DNA within the nucleus; and ethanol is used to precipitate the DNA. 4. What is the difference between the filtrate and the precipitate? During a precipitation reaction, resulting in the formation of a solid, after the precipitation, you use filtration to separate the liquid (filtrate) from the solid (precipitate). The solid collected on the filter paper would be the precipitate, and the liquid that passes through would be the filtrate. 5. Is there DNA in your food? How do you know? Why are you not harmed (or altered) by ingesting the DNA of another organism? Yes, we are eating the DNA and proteins of an organism when we consume and digest its cells. Because the digestive process is highly effective at breaking down and processing the components of ingested cells, and what our bodies absorb is primarily the basic building blocks like sugars, amino acids, fatty acids rather than intact DNA or proteins from the consumed organism. Part 2: Gel Electrophoresis using an Agarose Gel Gel Electrophoresis is a technique used to separate the DNA fragments according to their size and charge. They are placed on a sheet of gelatin (agarose gel) and an electric current is applied to the sheet. DNA is charged and will move in an electric field toward the positive pole. In the diagram below, holes (wells) in the agarose gel can be seen. DNA samples placed in these wells will migrate through the gel toward the + side after an electric current is applied. The smallest fragments will move the fastest because they are able to move through the pores in the gel faster. Bands will be produced on the gel where the fragments accumulate. The shortest fragments will accumulate near one end of the gel and the longer, slower- moving ones will remain near the other end. Materials:  Agarose Gel  Micropipette  Micropipette tips  Power source  Gel electrophoresis chamber  DNA samples

Figure 3: This diagram shows how to properly load the “DNA” into the wells of the agarose gel using your micropipette.  Place the cover on the apparatus and connect the red and black leads (red is positive, black is negative).  Notify your instructor at this point and they will turn on the voltage for you. The gel is run at ~90 volts for 20 minutes. Once the voltage is applied, observe that samples migrate towards the positive electrode (red) or negative electrode (black). Do not leave the gel until you are sure that the samples are migrating in the correct direction.  Turn off the power supply. Unplug the leads. Visualize the bands of dye which represent the DNA fragments. Draw your results.  Rinse gel equipment with water. Put gloves, paper towels in the regular trash. Dispose of electrophoresis buffer as instructed (you may recycle it, check with instructor). Wash hands. Results: Draw where your bands are located. Take note of the orientation of the gel going to the positive or negative side. Make sure to label the bands.

Conclusion: 6. What did you learn from this experiment? Understanding how to recognize and analyze DNA fragments of different lengths. 7. How can this technique be used? Provide two examples. Gel electrophoresis is used in genetic testing, it allows you to analyze the patterns by separating DNA by size when a segment of amplified DNA from a genome is digested with a restrictive enzyme. It is also used in forensics, to create DNA fingerprints from a crime scene and suspect samples, distinguishing between samples of genetic material. 8. Why did the DNA move the side of the gel that it did? The DNA fragments were drawn to the positive red electrode because it is negatively charged. 9. What is the purpose of the buffer? The TBE buffer allows for the current to take place, to flow through the sample. It provides ions that carry a current through the gel, to maintain a constant pH 10. Why was one band further down in the agarose gel than the other? Smaller DNA fragments move faster through the agarose, so they move farther in the gel than those that are bigger because the smaller fragments have less resistance in the agarose matrix. Licenses and Attribution:  " DNA Extraction – Strawberry " by Shannan Muskopf , LibreTexts is licensed under CC BY-NC-SA .  " DNA Extraction " by LibreTexts is licensed under CC BY-NC .  " Basic Techniques to Manipulate Genetic Material (DNA and RNA) " by Lumen Learning , LibreTexts is licensed under CC-4.0-BY .  " Gel Electrophoresis " by Ellen Genovesi, Laura Blinderman, & Patrick Natale , LibreTexts is licensed under CC BY .  " DNA Profiling " by LibreTexts is licensed under CC BY-NC-SA 3.0 US .