HOW-TO-'DO-IT A Visual Demonstration of DNA Preparation from

* RALPH L. KEIL, LAURA K. PALMER Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021

Unprecedented advances in unraveling mysteries of life have disease, or other areas the students or instructor want to develop. occurred as a result of the molecular biology revolution. Some Since many students know someone with diabetes (in many cases major achievements of this revolution include new methods for the they know a classmate who has diabetes or one of them may even production of therapeutic compounds, insights into mechanisms suffer from the disease), this example provides an opportunity to of gene function and regulation, and the complete sequencing of get students to actively participate in discussion, regardless of their the genomes of numerous organisms including humans. A critical academic abilities. foundation of this revolution is the ability to use the bacterium An alternative introduction is to discuss consequences of (E. coli) as a factory to produce large amounts of sequencing the entire three billion base pairs of DNA of the virtually any DNA of interest by inserting it into a small, extrachro human genome (http://www.ornl.gov/sci/techresources/Human mosomal circle of DNA called a plasmid. The ease and reproducibil Genome/home.shtml). The current and potential uses of this ity of plasmid DNA isolation from bacteria permits numerous labs technology in forensic science, health care, and business decisions to conduct molecular biology experiments and greatly accelerates are all areas that may be discussed depending on what focus the progress in understanding complex biological processes. instructor wants to develop. Many students who are not normally High school biology and introductory undergraduate biology interested in biological sciences become interested through dis or molecular biology classes frequently include labs in which stu cussions regarding possible uses of personal genetic information. dents either isolate or manipulate plasmid DNA. Such labs often Students are often surprised to learn that their ability to obtain consist of a "cookbook" lab protocol that contains little, if any, health or life insurance or their employment may be affected by information as to the purpose of the various steps involved in isolat the DNA sequences they have inherited (http://www.ornl.gov/sci/ ing the plasmid DNA. A major problem with this approach is that it techresources/Human_Genome/elsi/elsi.shtml). leaves the procedure as amysterious "black box" formost students. An additional means of generating interest is to have a culture To assist in illustrating the roles of the different steps of the proto tube with an overnight growth of E. coli.Asking the students if they col, amodel bacterial cell can be constructed with readily available have heard of E. coli normally elicits recounts of news stories of materials and used in a highly interactive presentation that visually food contamination. Discussion of differences between strains of E. demonstrates the purpose of each step. This demonstration can be coli that are pathogenic and non-pathogenic can lead to the revela used as a stand-alone lesson when introducing recombinant DNA tion that E. coli are normal inhabitants of our intestines that, along or it can be used as part of an introduction to awet-lab experiment with other intestinal bacteria, are necessary for us to live as they (a simple, rapid, low-cost, and highly reliable protocol is presented help digest our food as well as provide nutrients such as vitamin K. in the Appendix). The demonstration and the discussion related to (Some brave students may be persuaded to smell the tube of bacte it provide numerous opportunities to stress concepts included in ria,which normally produces interesting facial contortions.) the National Science Education Standards. O An E. coli Model To Illustrate Plasmid O Developing Student Interest Purification Why should I care? seems to be a common question in the minds of many students in introductory courses. Depending on the time Materials available, the level of the students or their interests, a number of * about twenty 3/8" flat metal washers from a hardware approaches can be used to help capture students' attention and store engage them in class discussion. In high school freshman biology classes, we found that a means of introducing recombinant DNA * about 2 m of twine (or string) with a double-helix structure technology and the role it can play in students' lives is to use an with the ends tied together to form a "circle" example of cloning a specific gene. Because of the prevalence of * quart-size zip-lock baggie diabetes, a useful example is the cloning of the human insulin gene * 6.5 by 9.5" manila envelope and the production of human insulin for therapeutic use in bacte ria (Ladisch & Kohlmann, 1992). This example permits student * enough marbles to fill the baggie involvement by questioning them about what they know about * 0.7 by 1m piece of carpet diabetes, the role of insulin in the disease, the health effects of this

THE AMERICAN BIOLOGY TEACHER PLASMID DNA PREPARATION i Construction of the Model E. cofi Cell students can be prompted to identify the twine as representing the bacterial (the double helical structure should be The washers (representing plasmid DNA molecules), twine very helpful) and the marbles as representing components of the (representing the bacterial chromosome of E. coli), and themarbles cytoplasm, such as enzymes or ribosomes. Use the scissors to cut (representing , ribosomes, and other components of the the twine into several pieces and explain that due to the extensive bacterial cytoplasm) are placed in the baggie (representing the length of the bacterial chromosome, it sheers when the plasma cell's membrane), which is then sealed. The filled baggie is then membrane is lysed. Once the twine is cut into pieces, the two inserted into the manila envelope (representing the bacterial cell strands can be pulled apart to demonstrate how DNA is denatured wall), which is then sealed. A single washer is retained to demon (separated into single strands) by the alkaline solution (due to strate what the students want to isolate and how difficult it is to the NaOH in Solution II). If appropriate, this provides a chance to locate the approximately 20 washers among the twine and marbles remind students about acids, bases, and pH as well as a chance to at the various stages of the protocol. discuss how double-stranded DNA can be denatured by alkali or Students can be provided with a copy of a protocol for plasmid heat. (One concept that can be difficult to convey is that the plas DNA isolation that has explanations for each step. The protocol mid DNA is also denatured by this treatment but since it is fairly in the Appendix is an example of an alkaline protocol that small and the two single stands are intertwined, they are still held has been very successful in high school freshman biology classes together and will renature [reanneal] when the solution is neutral ranging from "applied" to "honors." The demonstration can be ized in the next step.) Point out that it is still not easy to find and conducted as a "dry lab" demonstration or as a prelude to a "wet isolate the washers from the marbles and twine pieces. Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 lab" in which the students isolate plasmid DNA and conduct gel Next, "glue" is added (the high salt and potassium in Solution electrophoresis to assess their DNA yield. The model permits dis III; Step 7 of the Appendix) that binds to the proteins (marbles) cussion regarding what the various objects in the model represent. and denatured chromosomal DNA and the detergent from Solution This discussion can be expanded to talk about the composition of II. Spinning this mixture in a tube at high speed forces all the com any of these components, depending on the learning objectives ponents that have been "glued" together to the bottom of the tube appropriate for the class. (The discussion also permits correction (Step 9; Appendix). This can be simulated by gently pushing the of students' misconceptions, e.g., in our experiences students have suggested the marbles represent mitochondria, although organelles do not exist in bacteria). Figure 1. Visual demonstration of plasmid isolation from "Dry Lab" Plasmid Purification E. coli. A) intact cell B) removal of cell wall C) plasma membrane lysis Demonstration and release of cellular contents D) removal of cellular debris E) plas A convenient way to begin the demonstration is mid concentration. See text for further details of each step. by holding the model E. coli cell (Figure 1A) in one hand and a washer ("plasmid") in the other hand and ask the class if it could easily isolate (or even see) the 20 in the bacterial cell. Ideas as towhat component of the bacterial cell is represented by the manila envelope can be solicited from the students. Their identification of it as the cell wall provides an opportunity to discuss the fact that cells of some organisms like bacteria and plants have cell walls while our cells do not. Further discussion on why that is the case is certainly recommended. Scissors can be used to cut through the manila envelope (being care ful not to cut the underlying baggie; Figure 1B) while explaining that it is first necessary to get through the cell wall (accomplished by Solutions I and II in Steps 5 and 6 of the protocol in the Appendix). Holding the baggie, show students that it is still very difficult to locate and isolate the plasmids. Students can be questioned as to the cellular com ponent represented by the baggie until someone says the cell membrane. They can then be pressed regarding the components of the cell membrane until lipids (or phospholipids) are named. By introducing the concept that lipids are similar in composition to grease, oil, and fat, the students should be able to suggest that a way to get through the membrane is to add detergent. This can be likened to someone remov "plasmidis" ing an oil spot from clothing. Scissors can be used to cut the baggie (or "lyse the membrane"; accomplished by the SDS detergent in Solution II in Step 6 of the Appendix). Pour the marbles, twine, and washers out near one edge of the piece of carpet (Figure IC). The

THE AMERICAN BIOLOGY TEACHER a VOLUME 71, NO. 6, AUGUST 2009 marbles and twine to the other edge of the carpet with your hand. (Answer: Intact plasmids are circular and "supercoiled,"mean This completely separates these components from the plasmids ing that the double strands of the plasmid twist around each (Figure ID), which don't move when the marbles and twine are other. This can be demonstrated by holding a rubber band at being pushed. two distant sides and twisting the rubber band into a figure The washers remain quite spread out on the carpet at this eight [representing one supercoil] or continuing to twist to intro point. The precipitation of plasmids in alcohol followed by centrifu duce additional supercoils. These supercoiled forms generally gation can be demonstrated by using fingers to push the washers runfaster than linearfragments that contain the same number to the side of the carpet away from the marbles and twine (Figure of DNA basepairs.) IE; Steps 10 and 11 of the Appendix). As an alternative to alcohol For advanced students: Indeed, intact plasmid DNA often precipitation for concentrating plasmid DNA, many labs use DNA runs as several distinct bands on a gel, often three or more. purification columns to accomplish this. These columns contain a Give explanations that account for finding three different resin that selectively binds plasmid DNA that can later be released bands from one intact plasmid. by passing water through the resin. To demonstrate column purifi (Answer: There are a) supercoiledforms of the plasmids, which cation of plasmid DNA, amagnet can be used to collect the washers are the normalforms in bacteria, as well as b) linearforms that that are then removed from the magnet by hand. result from breaks in both strands of DNA that are inadver O Extensions: Fun with Big & Small tently introduced during plasmid preparation, and c) "relaxed" circularforms an untwisted rubber band] that resultfrom [like Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 Numbers a break in only one strand of DNA during the isolation proce It can be enlightening for students to discuss some very large and dure.) very small numbers that are related to this demonstration and 3. If you could clone, sequence, and manipulate any one gene experiment. For example: from any organism, what gene would you want to clone and 1. In a 1.5 ml overnight culture of bacteria (a standard volume from what organism? Why? of culture used tomake small amounts of plasmid DNA in 4. In the protocol in theAppendix, Step 5 involves resuspend a lab), there are more bacterial cells (about 7.5 x 10') than ing the cells in 100 p1 of Solution I.What fraction of a liter there are people in the world (about 6.6 x 109 as of 6/22/07; of soda is 100 pl? (Answer: 1/10,000) http://www.census.gov/ipc/www/popclockworld.html). 5. Molecular mass of base pairs or plasmid is normally 2. A typical E. coli cell is about 1.4 pm long while the bacte expressed in Daltons (Da) since their weight is very small rial chromosome is about 1,400 pm in circumference; that (one Da is approximately 1.7 x 10-24grams). An average is, the chromosome is 1000 times as long as the cell. To base pair has amass of 650 Da. What is themolecular mass make a scale model using a manila envelope that is 24.1 of a single copy of a 5 kb plasmid DNA in Daltons? (Answer: cm (9.5 inches) in length to represent the bacterial cell, it 3.25 x 106Da) would be necessary to use a string that is 2.41 x 104 cm (i.e., 241 m or 9,500 inches, 792 feet or 264 yards) long. This In grams? (Answer: 5.53 x 10`8g) "Cstring"would have to be incredibly thin if it were also to 6. Following isolation of a 10 kb plasmid from E. coli, it is scale-about 3.55 x 10-5cm or 355 nm (0.000014 inches) in estimated that the total yield of plasmid DNA is 1 pg. How diameter (DNA is 20 A in diameter and one A [angstroms] many copies of the plasmid are present in the tube? is 100 x 10-12m, 0.1 nm, or 9 x 10' inches in diameter. For comparison, a human hair is about 1,000,000 A in diam (Answer One plasmid would weigh approximately 11 x 10-'2 eter.) pg. Therefore, 1 pg of plasmid DNA would have approximately 9 x 1010copies of the plasmid. This is 90 billion copies of the 3. A "typical" plasmid contains about 5,000 base pairs (bps) plasmid.) or 5 kilobase pairs (kbs), the E. coli chromosome contains about 5,000,000 bps (5,000 kbs), and the human genome 7. Why might a potential employer be interested in knowing contains 3,000,000,000 bps (3,000,000 kbs or almost 1 if someone has a particular gene sequence that increases million times the amount of DNA in a "typical" plasmid). her/his chances of developing cancer? (Answer: A variety of reasons including lost productivity due to inability towork dur O Assessment & Discussion Questions ing treatments and associated illnesses.) Assessment or further discussion can explore a variety of different Do you think the employer should have the right to use areas including: knowledge of biology; links between biology and personal genetic information when making a decision on mathematics; or ethical, legal and social issues. Sample questions whom to hire? Why or why not? Who should determine in these various areas include: this? How should it be regulated and enforced? 1. these are double if a 5 kb Remembering plasmids stranded, 8. If you could have your DNA sequenced for $50,000, would has 2300 guanine (Gs) how many cytosines plasmid bases, you want to have it done? Why or why not? (Would your (Cs) does it have? (Answer: 2300) answer change if the cost were only $5,000?) If they found How many adenines (As) does it have? (Answer: 2700) you had a mutation in your DNA that increased your risk How many thymines (Ts) does it have? (Answer: 2700) 50-fold of dying from some incurable disease by the time you were 45, would you want to know? How do you imag 2. During gel electrophoresis, linear fragments of DNA sepa ine this would change the way you lived? rate according to size. However, intact plasmid DNA does not run at the same position as DNA from the same plas Additional ideas, questions, and links regarding ethical, mid after it has been linearized by digestion with a restric legal, and social issues can be found at http://www.ornl.gov/sci/ tion enzyme that cuts it once. Why? techresources/ Human_Genome/ elsi/ elsi. shtml .

THE AMERICAN BIOLOGY TEACHER PLASMID DNA PREPARATION a O Addressing National Science APPENDIX. Protocol for Plasmid DNAMini-Prep from Standards Bacteria This demonstration supports several facets of the National 1. Grow bacterial culture in 2 ml of medium with appropriate antibiotic over Science Education Standards (National Research Council, night. 1996). One of the major goals of the Life Science Content 2. Add 1.5 ml culture tomicrocentrifuge tube. Standards for grades 9-12 is to expand students' under 3. Spin in centrifuge for one minute atmaximum speed. standing of biology by "incorporating more abstract knowl edge, such as the structure and function of DNA" into the 4. Pour off media. curriculum, as well as to solidify the concept that "cells have 5. Resuspend pellet in 100 p1Solution I. particular structures that underlie their functions." In addi 6. Add 200 p1Solution II. tion, this lesson fits well within the vision for the Standards 7. Add 150 p1Solution III. for Science Teaching (National Research Council, 1996). 8. Mix briefly by vortexing. The lesson: facilitates inquiry-based student learning by 9. Spin in centrifuge for threeminutes atmaximum speed. capitalizing on interests, knowledge, and abilities appropri ate for the grade level (Teaching Standards A and E); allows 10. Add supernatant to 1ml of 95% . Vortex tomix. teachers to guide and facilitate student learning through a 11. Spin in centrifuge for one minute atmaximum speed. series of directed question-and-answer sessions relating the 12. Pour off ethanol. laboratory to "big-picture" concepts students encounter in 13. Invert open tube on paper towel to dry.When pellet is dry, resuspend pellet Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 their daily lives (Standard B); provides ameans to bring the in 200 p1TE (10 mM Tris pH 7.4 and powerful technology associated with current molecular bio 1mM EDTA pH 8.0 with 0.2 mg heat-treated RNase A per 10 ml TE) by vor logical applications to high-school aged students (Standard texing. D); and actively engages students in collaborative exercises 14. Put 20 p1of this DNA solution into the microcentrifuge tube containing 4 p1 in both lecture and laboratory settings (Standard E). of purple dye (0.1% bromophenol blue in 30% glycerol). Mix. This lesson also focuses on skills required for success 15. Load 10 p1of the DNA/dye solution onto a 0.8% agarose gel along with 10 in entry-level undergraduate science courses as detailed by p1 of the X size standard/dye solution. the report, Understanding University Success (AAU, 2003). 16. Run gel at 150 volts until blue dye is approximately 1/2 way down the gel. This report summarizes a comprehensive study of con 17. Put on a UV transilluminator and photograph. (Do NOT look at transillumi tent knowledge and abilities high school students should nator without safety glasses.) master to succeed at the university level. For the Natural Sciences, these standards include: "General Foundation Skills," such as knowing basic math conventions and under Solutions standing and applying concepts of measurement; "Science SOLUTIONI SOLUTION11 SOLUTIONIII and Society" standards, such as understanding how science 50 mM glucose 2 mL I N NaOH 60 mL 5 M KOAc and society influence each other in terms of technological advances, funding, and public policy; and "Biology"-specific 25 mM TrisHCI, pH 8 1mL 10% SDS 11.5 mL glacial standards, including understanding the general structure acetic acid and function of cells. 10 mM EDTA,pH 8 7mLH20 28.5 mL H20 O Summary Autoclave and store at 4 'C. This lesson provides an engaging, low-cost, visual dem onstration linked to interactive classroom discussion that Explanations teaches fundamental concepts underlying the molecular Step 1: Selective medium with an antibiotic is used to maximize the amount of biology revolution. The visual demonstration and discus plasmid in the culture. (Only cells with the plasmid can grow in medium sion develop student interest and assist their learning of the containing the antibiotic.) concepts. A rapid, reproducible wet-lab exercise in which Steps 2 through 5: Remove the growth medium and resuspend the cells in a buf students isolate plasmid DNA and visualize it by gel electro fer (Solution I). phoresis can be added if resources and time are available. Step 6: Solution II breaks open (lyses) the cell. SDS is a detergent used in science (and in household products such as shampoo) that dissolves the cell mem References brane and NaOH is a strong base that denatures cellular proteins and DNA.

Association of American Universities (AAU) & The Pew Charitable Steps 7 through 10: Solution III is an acidic, high salt solution (students often Trusts. (2003). Understanding University Success. A Report from recognize that it smells like vinegar, which is a dilute solution of glacial acetic Standards for Success. Eugene, OR: Center for Educational Policy acid) that precipitates the cellular , denatured chromosomal DNA, Research. Available online at: http://www.sHs.org/. and the SDS detergent. The white material in the tube is protein, lipid, bro Ladisch, M. R. & Kohlmann, K. L. (1992). Recombinant human insulin. ken DNA, and detergent that form insoluble complexes with components of Biotechnology Progress, 8(6), H69-H78. Solution III.This will form awhite pellet when it is centrifuged. The plasmid National Research Council. (1996) National Science Education DNA renatures when the alkali of Solution II is neutralized by the acid of Standards. Washington, DC: National Academy Press. Available Solution III and the plasmid DNA will not pellet. online at: http://books.nap.edu/readingroom/books/nses/html/. Steps 11 and 12: Adding the supernatant to the ethanol precipitates the plasmid DNA. DNA is very soluble inwater, but not in ethanol. Step 13:When centrifuged, the DNA will form a clear pellet at the bottom of the o RALPH L.KEIL, isAssociate Ph.D., Professor, Department of Biochemistry tube (much smaller than the white protein/lipid pellet). c and Molecular Biology, Penn State Hershey Medical Center, Hershey, PA 17033-0850; e-mail: [email protected]. LAURA K. PALMER,Ph.D., isAssistant Steps 14 and 15: After the ethanol dries, the DNA can be dissolved in TB. (TB is Professor of Biology, Division of Mathematics and Natural Sciences, a solution that ismostly water, but contains chemicals that bind Mg'+, an ele Penn State Altoona, Altoona, PA 16601; e-mail: [email protected]. ment needed forDNA-degrading enzymes.)

a THEAMERICAN BIOLOGYTEACHER VOLUME71,NO. 6,AUGUST 2009