> Teacher’s Manual DNA Extraction Lab Activity W56615 38 Scientific GmbH, Rudorfieg & + 21031 Hamburg ~ Germs Tol: 40-407 pac 4 40-40-72088-100 Sbeciartife.cem + Ab@abecienis. com“~ 2 DNA Extraction Lab Acti WS56615 Be INTRODUCTION In 1869, a Swiss scientist by the name of Friedrich Miescher isolated an unknown substance from white blood cells. It wes a white, stringy, slightly acidic substance that Miescher eventually termed “nuciein” because it seemed to be found in the nucleus of the cells. Miescher also determined that the material was rich in phosphate-containing substances. Earlier in the same century, an Austrian monk by the name of Gregor Mendel was exper- imenting with pea plants. Mendel was examining not only certain characteristics (traits) of the plants (such as the height of the plant, flower color, and color of the pea the plant produced) but also what happened to these characteristics when the different pianis were crossed with each other to produce new plants. Mendel concluded thal the new plants sometimes showed traits from one of the original parent plants. In crossing plants that produced green pea seeds with planis that produced yellow pea seeds, Mendel showed that some of the offspring plants might have green seeds and others might have yellow pea seeds. Even more importantly, Mendel noticed that the crosses consistently produced around the same number of offspring with each trait. Maybe the offspring would all show the trait of one of the parent plants, maybe about half of the offspring would have a trait from one parent and about half would have a trait from the other par- ent, maybe for every three plants that showed one trait, one would show the trait from the other parent, etc. Mendel also noted that in some cases, the offspring displayed what appeared to be a combination of the traits from each parent. For example, by crossing plants that produced dark purple flowers with plants that produced white flow- ers, Mendel was able to produce offspring with pink or light purple flowers, seemingly a combination of dark purpie and white. Mendel’s work was largely ignored during his lifetime. Its impact was not fully realized until well after his death and would eventually come to be the basis of modem genetics. Likewise, the full importance of Miesher's discovery was not fully appreciated until after his death. While several scientists continued to experiment with Miescher's ‘nucicin,” it was not until decades later that it would come to be known as deoxyribonucleic acid, or DNA. Further research would also show that not only was Mendel correct, that traits passed to offspring were determined by the traits contained in parent organisms, but also that the information that controlled these traits was contained in DNA. Copyignt 2012 Gateway Chemicals Lis ‘www. Sbscientific.comDNA Structure During the first half of the twentieth century, new analytical techniques in the field of chemistry were being developed at a rapid rate. By mid-century, scientists had deter- mined exactly what chemical compounds were found in DNA. As it turned out, DNA was. constructed from just a few components. While DNA is a very large molecule, it is also a relatively simple molecule. For this reason, most scientists refused to believe that DNA was the material responsible for genetic inheritance and genetic diversity. Surely there was no way that that such a simple substance could account for the huge variety of dif- ferent life forms, let alone the large variety of different traits found within members of the same species. At the time, proteins, by far much more complex substances, were con- sidered the more likely source of the transmission of genetic information While the materials that composed DNA were well known, it would not be until the meth- od in which these materials were assembled, the actual form of DNA, was determined that it could be shown that DNA was indeed the basis of genetic information. In 1953 two scientists working at Cambridge University, James Watson and Francis Crick, were able to develop the first model of a DNA molecule. It was this groundbreaking insight into the structure of the material that revolutionized the thought and approach to genetics asa science. So important was this discovery that Watson and Crick were awarded the Nobel Prize a few years later. It should be noted that a great deal of research was conducted on DNA during decades previous to Watson and Crick's work. Some microbiologists were conducting experi- ments indicating that DNA could be the basis of genetics. Some scientists were using new techniques involving x-rays to help determine the structure of many chemicals, including DNA. In many ways, it was almost as if the pieces of the puzzie were out there 5 and Watson and Crick assembled the puzzle to see the resulting picture. ‘As mentioned above, Watson and Crick’s discovery revolutionized the field of genetics. It was this insight into structure that helped determine exactly how DNA could function as the basis for genetic information. 1t also led to what has become known as the “central dogma” of molecular biology. By understanding the structure of DNA, Watson and Crick were able to explain how DNA molecules could contain the code, or blueprint, for the eventual formation of proteins. In their model, Watson and Crick showed that a DNA molecule was a double helix. It contained two long strands twisting around each other. The strands are composed of linked molecules called nuciectides. A nucleotide is composed of three basic subunits, a 5.carbon sugar called deoxyribose, a phosphate group, and a nitrogen base. For each strand of the DNA molecule, the phosphate group of one nucleotide links to the deoxy- ribose portion of the next nucleotide. The nitrogen base of each nucleotide is attached to the deoxynbose and sticks off of a different part of the sugar. So the length of each strand of the DNA molecule is actually a chain of alterating phosphate and deoxyribose www. 3bscientific.com Conyigh2012 Gateway Chemicals Lidmolecules, while the nitrogen bases are not actually incorporated into the chain. Itis the fact that these nitrogen bases emerge from another part of each nucleotide, however, that gives such a simple molecule such incredibly complex function. Nucleotide Structure Phosphate Group Deogritore Nitrogen Base There are only four different nitrogen bases that can be attached to each nucleotide from which DNA is formed. This is one of the reasons that many early researchers refused to believe that DNA could be responsible for the transmission of so much complex informa- tion. The four different nitrogen bases are called adenine, cytosine, guanine, and thy- Mine (often referred to in shorthand as A, C, G, and T). Previous research had revealed another piece of information: in a DNA molecule, there were always equal amounts of adenine and thymine and there were always equal amounts of cytosine and guanine. This led to the conclusion that in a DNA molecule, an adenine (A) base must always be paired with a thymine (T) base and a cytosine (C) base must always be paired with a guanine (G) base. In Watson and Crick’s model, they showed how this would work. Both strands of the molecule (composed of the sugar and phosphate) twisted around each other with the nitrogen bases sticking off and linking the two strands together. Every adenine on either strand was linked to a thymine on the other strand and every cytosine on either strand was linked to a guanine on the other strand. The resulting molecule, according to Wat- son and Crick, resembled a spiral staircase with the sugar-phosphate strand forming each edge of the staircase and the linked nitrogen bases forming the steps. Watson and Crick’s model also explained how DNA could self-replicate (copy itsetf), With their model, the two strands of the DNA molecule could separate from each other to form two single strands. Each strand would have its nitrogen bases exposed since Coppigh2o'2 Calenay Chemicals Lid. www 3bscientific.comthey were no longer paired to the other strand. New nucleotides could attach along each exposed strand. Since the nucleotides always pair up in a specific fashion, each single strand vould lead to the formation of a new strand, absolutely identical to the strand that separated from it earlier. Functions such as growth and tiscue damage repair are accomplished through cell division. Existing cells divide to form now cells in a process called mitosis. Using this model, it can be seen that DNA in the nucleus of a cell could replicate itself and provide an identical copy used during the formation of a new cell. It also showed how DNA could contain the information for complex genetic traits. DNA Function and the Central Dogma At one point it was believed that proteins were responsible for the passage of genetic information. While this is not true. proteins are responsible for the expression of genetic information. For example, eye color or hair color in a human results from the presence or absence of certain proteins within the individual. Whether or not an organism will or will not produce these proteins is determined by the combination of genetic information which is inherited, some of this information from one parent and some from the other Parent. The key to understanding this inheritance is knowledge of the term “genetic infor- mation.” Using the human example again, an individual will not display a certain eye color because of proteins inherited from each parent. What is inherited is the informa- tion for the individual to make these proteins, and these proteins will eventually result in eye color. The information to make these proteins is contained in DNA. ‘The move from DNA to protein involves another nucieic acid known as ribonucleic acid (RNA). RNA is similar to DNA in that it is constructed from four nitrogen bases, but there are a few notable differences. Firstly, the base thymine is not present in RNA. Instead, RNA has a base called uracil (U) while the other three bases remain the same (A, C. and G). Secondly, RNA contains the sugar ribose, instead of deoxyribose thal is found in DNA. Lastly. unlike the two twisting strands of DNA, RNA has a single strand and the < molecules tend to be much smaller than DNA molecules. ‘The central dogma states that: DNA —» RNA—» Protein In other words, DNA leads to RNA and RNA leads to protein, In the nucleus of a call, the DNA strands separate from each other. Special enzymes construct RNA molecules using the information from the DNA. As the enzymes move along the single strand of DNA, cytosine (C) is always paired with guanine (G) and vice versa. Adenine (A) is always paired with any thymine (T) found on the DNA strand. When the forming RNA molecule encounters an adenine on the DNA strand, it uses the base uracil (U) instead of thymine. The smaller RNA molecules are able to pass through the membrane surrounding the nucleus of the cell. There, they encounter structures called ribosomes. The ribosomes are the sites at which the proteins are assembled. Different enzymes read the sequence of the RNA bases. This sequence instructs the ribosomes to add molecules called amino acids. Much like the four nucleotides (A, C, G, and 7) are the building blocks of DNA, amino acids are the building blocks of proteins (but there are more than four www 3oscientific.com CCopynight 2012 Gateway Chemicals Lidamino acids. Itis the order in which amino acids are assembled that decides what the resulting protein will be. While this is a simplified version of the production of proteins from DNA, it give the basic ideas and shows how Watson and Crick’s work was able to not only show once and for ail that DNA is indeed the molecular basis of genetics and heredity, but also how DNA served this function Isolation of DNA ‘As DNAis considered the set of genetic ‘instructions’ for all living organisms, it stands to reason that all living organisms contain DNA. Using a few basic lab techniques, as, well as the right materials, this DNA can be isolated. The first step is to get the DNA exposed. DNAs found in the nucleus of cells, which is surrounded by a membrane, The cell itself is also surrounded by a membrane, and in the case of plants, a cell wall In order to eliminate these barriers, special biological detergents are added to the sam- ple. The detergent is mixed with the sample. The detergent “pops” the calls (a process called cell lysis). This destroys the call and nuclear membranes, releasing the DNA Unfortunately, cell lysis not only releases the DNA, but also releases other materials from the cells. Proteins, enzymes, and other cell structures are released into the de- tergent solution. One of the enzymes released is called DNase. Enzymes are special forms of proteins that serve a variety of functions. DNase is an enzyme that breaks up DNAmolecules. Trying to isolate DNA would be complicated by the fact that there is, an enzyme in the sample that is trying to destroy the DNA. For this reason, a different enzyme, called a protease, is added. Proteases are enzymes that break down pro! There are many types of proteases. Since an enzyme is a form of protein, a protease will break down enzymes, including DNase. Once the DNAis released from the cells through the action of the detergent and the DNAis protected from DNase through the action of a protease, the sample is filtered to remove any remaining solid material left from the original sample. The solution that re- sults after mixing still contains a great deal of material other than DNA. In order to get at the DNA, a small amount of the alcohol ethanol is added to the filtered solution, DNA is not soluble in ethanol, especially very cold ethanol. When added to the filtered sample, ‘many of the other contaminants from the cell stay in the solution but the DNA precipi- tates out. In this basic procedure, the DNA that precipitates out will still have some contaminant maierial. In an actual biotechnology lab, further purification steps would be performed to purify the DNA more. However, using these steps, DNA can be isolated and removed from practically anything! ConyeghZ012 Gaveway Chemicals Lt vaww.3bscientific.com.eT Objectives + Leam the history of the discovery of DNA and DNA structure. + Understand the nature of genetic inheritance and the role of DNA and proteins in genetic expression + Use biological detergents, enzymes, and ethanol to isolate DNA from plant or animal material. Materials Included in the Kit ~ 2° 7.5% SDS/1.5% NaCl, 250ml (10 1 Pepsin (to make 25ml 0.5% solution) 2 $5% ethanol, 25m! 20 Zipper bags 15 Filters 15 Plastic tubes 30 Graduated pipettes 15 Stirrers Materials Needed but not Supplied DNA source (2.9. fruit or vegetable material) Graduaied cylinders, to measure 25ml - Funnels ‘Small beakers or plastic cups Ice bath or freezer Distilled or deionized water Optional (depending on DNA source): Mortar and pestie -or Laboratory blender Safety Gloves Safety goggles Lab Apron www.3bscientific.com Copyright 2012 Gateway Chemicals Lia aPre-lab Preparation Prepare the 0.5% pepsin solution 1. Just prior to performing the lab, add 25m! of distilled or deionized water to the bottle containing the pepsin powder. 2. Cap tightly and shake until completely dissolved, Prepare cold ethanol 1. Prior to performing the lab, prepare cold ethanol by either placing the bottles of etha- nol in an ice bath on the lab bench or placing the bottles of ethanol in a freezer. Instructor's Notes There are many suitable sources of DNA for isolation. Soft fleshed fruits such as peeled bananas, strawberries, blueberries, or peeled Kiwis are ideal to work with and contain a great deal of DNA. The procedure for this activity is presented in such a way that stu- dents will piace the material in a sealed bag and physically mash the tissue in the deter- genlenzyme mixture, A few grams of material (such as a 1 centimeter slice of banana, Couple of slices of a large strawberry, a couple of blueberries, etc.) should be sufficient for extraction. Other materials such as onions, raw (not toasted) wheat germ, peas, and beef liver also serve as a good source of DNA. However, using materials such as this would require the use of a mortar and pestle or laboratory blender to macerate the material enough to ensure successful DNA isolation. Always use fresh, not cooked or processed, material. When exposed to cool or cold temperatures, such as those that may occur during ship- Ping, the 7.5% SDS/1.5% NaCl solution may develop a white precipitate in the bottie. Do Not use the solution with the precipitate present. Slightly warming the material will al- ‘ow the precipitate to easily go back into solution. You may place the boitle(s) in a warm (does not need to be hot) water bath, microwave the boitle(s) in 30-second intervals (be Sure to loosen the cap(s) prier to placing in the microwave) until the precipitate disap- pears. or warm the solution by holding the bottle(s) under hot running tap water, Conyigh2012 Gateway Chemicals Li www.3bscientific.comProcedure Materials Needed per Group Zipper bag Graduated pipettes Filter Plastic tube Stirrer Small beaker or plastic cup Funnel ~ Shared Materials aaone 7.5% SDS/1.5% NaCl solution 0.5% pepsin solution 95% ethanol Safety Gloves Safety goggles Lab Apron 1. Place a few grams of a DNA source, assigned by your instructor, in your zipper bag. 2. Using a graduated cylinder, measure out 25ml of 7.5% SDS/ 1.5% NaCl solution. 3. Pour the measured solution into the bag containing your DNA source. 4. Using one of your graduated pipettes, add 1ml of 0.5% pepsin solution to the bag. 5. Squeeze the bag to remove as much air as possible, without losing any liquid or sample, and seal the bag. 6. Completely macerate the DNA source using your fingers (wearing gloves) to mash the material into the detergent solution. The more thoroughly the sample is mixed with the detergent, the more DNAwill be released. Be sure to use enough force to macerate the tissue but not $0 much force as to cause the bag to leak. Continue for a minimum of five minutes (firmer material may require more time). 7. After the sample is thoroughly mixed, set aside and place your filtor into a funnel. Place the funnel over your small beaker or plastic cup. rw 3bscientific.com Copyright 2012 Gateway Chercal Lic a8. Open your sealed bag containing the DNA/detergent solution and carefully pour the contents into the filter inside of the funnel. 9. Allow the liquid to completely filter into the beaker or cup. 10. Pour approximately 2-3ml (about 3em depth) of the filtered solution into your plastic tube. 11. Using your other graduated pipette, add 2m! of cold 95% ethanol to the solution in the tube. Try to squirt the ethanol into the tube with enough force to mix it into the liquid in the tube. 12. After the cold alcohol has been mixed with the DNA/detergent solution, observe the contents of the tube. Continue to observe for a couple of minutes. 13. Using your plastic stirrer, reach into the tube and try and “snag” some of the DNA that formed in the tube. Once you have gotten some of the material, pull it from surface of the liquid. Ob- serve the properties of the DNA. Try touching some of the DNA to the inside of the tube and Pulling it away from the side. Try spooling the material by gently twirling the stirrer. Record any observations you make in the Data Analysis section of the lab. 14, Clean up and dispose of all materials according to your instructor. Be sure to wash your hands before leaving the lab Copyight2ot2 Gateway Chemica Lt, ‘www 3bscientific.com