The DNA Discovery Kit The Discovery Approach & Teacher Notes

Transcription

1 ...where molecules become real TM The DNA Discovery Kit & Teacher Notes All rights reserved on DNA Discovery Kit. US Patent 6,471,520 B1 Photos by Sean Ryan

2 The DNA Discovery Kit Contents of Contents of the DNA Discovery Kit 12 Base Pair Kit Contents of the DNA Discovery Kit 2 Base Pair Kit Assembly Instructions Optional Questions and Activities Reviewing Bonds Watson and Crick DNA Papers Contents of The DNA Discovery Kit 6 each of Adenosine, Thymine, Guanine and Cytosine Nucleotides 48 Nucleotide Labels 2 Mini-Toobers Black Helix Guide Contents of The DNA Discovery Kit 1 Each: Adenosine, Guanine, Thymine and Cytosine Nucleotides 8 Nucleotide Labels Mini-Toober DNA Three Frequently Asked Questions The Discovery of the Structure of DNA..17 Student Handout Transparency Templates Chemical Structures Information Available to Watson & Crick in Base Pair Kit Black Rod Black Base Assembly Instructions 2 Base Pair Kit Assembly Instructions Contents of Online Resources found at 3dmoleculardesigns.com/resources.php Contents & Introduction PDFs DNA Discovery Kit Introduction DNA Contents & Assembly Directions DNA Activities & Teacher Notes PDFs The Guided Discovery Approach Student Handout Three Frequently Asked Questions DNA Resource Information Read Me First Teacher Notes Student Worksheet Student Answer Sheet DNA Websites Additional DNA Resources 2 Watson & Crick Papers PDFs Watson & Crick April 1953 Watson & Crick May 1953 Annotated Version of Watson & Crick Paper

3 The DNA Discovery Kit DNA Discovery Kit Assembly Instructions Nucleotides Assembled The nucleotides are preassembled. You have the option of using labeled or unlabeled nucleotides. To label a nucleotide, peel a letter from its protective backing and press it into the depression on a corresponding base. After placing the label on one side, flip the base over and repeat with another label. Use the photo to correctly place the labels on the nucleotides. (Labels only fit inside the larger depression on the Adenosine and Guanine nucleotides.) Magnets Simulate Bonding Phosphodiester Bond Magnets The nucleotide models have magnets embedded in them to simulate the spontaneous bonding that occurs between complementary base pairs (hydrogen bonds) and between the phosphate group of one nucleotide to the deoxyribose of another nucleotide (phosphodiester bonds). Arrows in the photo above point to the magnet(s) in each piece. Hydrogen Bonds You can break the hydrogen bonds by pulling apart the G-C and A-T base pairs. When examining the deoxyribose and phosphate groups, you will see the single magnet embedded in the deoxyribose group and one embedded in the phosphate group. 3

4 The DNA Discovery Kit Nucleotides Separate Into Component Parts Each nucleotide separates into its three component parts the nitrogenous base, deoxyribose group and phosphate group. To separate the pieces, pull the three pieces apart as shown in the photos. Be sure to pull the pieces apart with a straight motion. The attachment posts can break if a twisting or bending motion is used. Three Ways to Display DNA Discovery Kit We encourage you to leave the DNA Discovery Kit pieces out on a table for your students to explore in their free time. You can also easily display or store the fully assembled double helix by setting up the black base and black rod that are included in the 12 Base Pair DNA Discovery Kit. Or you can hang the double helix from a ceiling by threading a strong cord through the eyelet at the top of the rod. Do not use the black base when hanging the DNA. 4

5 The DNA Discovery Kit Setting Up Base & Rod to Displaying DNA Eyelet for Hanging DNA on Rod Push the bottom of the rod (end without the eyelet) into the black base. Press down firmly so it rests securely in the base. The lowest disk will rest about 3/4 inch above the base. Before correctly placing the Guanine - Cytosine base pairs around the rod, look carefully at the models. You will see that the Guanine model has two hydrogen that are longer than the third hydrogen. (See photo at right and refer to the page 2 photo labeled, Hydrogen Bonds.) The Cytosine model has one hydrogen that is longer and two shorter hydrogen. Adenosine and Thymine each have one longer hydrogen and one shorter hydrogen. The Guanine - Cytosine base pair should be placed so that the rod is between the longer and the shorter hydrogen. (See the above photos.) The Adenosine - Thymine base pair should be placed so that the rod is between the two hydrogen (one is longer and one is shorter. As each base pair is placed on the rod, rotate it until it forms the phosphodiester bond with the previous base pair. Four base pairs fit above each disk. Making Mini-Toober DNA Place two Mini-Toobers side-byside, so the red end cap on one is even with the blue end cap on the other. Next, line them up with the first two grooves in the black helix guide. Begin wrapping the Mini- Toobers around the guide following the grooves. Once the Mini-Toobers are wound around the guide, you can remove them by twisting the guide as though you are unscrewing it from the Mini-Toobers. Then loosen and separate the coils by gently unwinding and pulling them apart. (See the Activities and Teacher Notes at 3dmoleculardesigns.com/resources.php for information on this activity.) Black Helix Guide 5

6 Teacher Notes to investigating the structure of DNA allows students to discover the double helix in much the same way as Watson and Crick discerned it in In addition to gaining an understanding of the structure, this approach enables your students to experience science as a dynamic, creative process, in which groups of people work together or in competition to construct an understanding of the natural world. The story of the discovery of DNAs double helical structure is a compelling example of real science. For this approach, evenly divide the bases, deoxyribose sugars, and phosphate groups among your students. Then pass out copies of the Student Handout, which contains the information that was available to James Watson and Francis Crick in This includes the polymeric nature of DNA, Chargaff's rules, and the crystallographic data of Maurice Wilkins and Rosalind Franklin. Then challenge your students to develop a model for the structure of DNA that satisfies the information available in Your students will quickly see: How the phosphate groups, the deoxyribose groups and the bases join together to form four nucleotides, How the complementary bases go together via hydrogen bonding to form the AT and GC base pairs How the base pairs stack upon each other to create the elegant double-helical structure of DNA that is described in Watson and Crick's 1953 publication in Nature. The Student Handout begins on page 17. The Information Available to Watson and Crick in 1953 appears in the blue box on page 2 of the Student Handout (page 18 in this document). (A summary of key points of the Information Available to Watson and Crick in 1953, (page 21), can be printed on transparency film for overhead use. The Student Handout is also available as a separate document on the website.) Phosphates Deoxyribose Nitrogenous Bases Hand out the 12 DNA pieces shown above (four phosphate groups, four deoxyribose groups, and the four nitrogenous bases, A,G,T and C), to each small group of students. Once each group of your students has the pieces, you may want them to refer to their last page of the Student Handout (see page 19) which identifies: the phosphate groups, deoxyribose groups, nitrogenous bases and the coloring scheme. Then tell your students to play with the pieces until they discover how all fit together in a compact structure. 6

7 Teacher Notes 1 2 One of the many possible ways to put together two base pairs of DNA (a GC and an AT base pair) is shown: Photo 1: The phosphates are joined to the deoxyribose groups. Photo 2: Sugar-phosphate backbone of DNA is discovered. This polymer contains no information. How do we know that it contains no information? Photos 3, 4, and 5: The bases (A,G,T and C) are added to the backbone. 6 Photo 6: Tell your students to unassemble the single-stranded DNA chain, so that they have four nucleotide units. 7

8 Teacher Notes Tell your students to play with the four nucleotides until they discover how The AT and GC base pairs fit together. Once each student group has put together their AT and GC base pairs, the base pairs can be combined to form the 12 base-pair double helix (if you purchased the 12 Base-Pair DNA Discovery Kit ). The DNA can be assembled horizontally on a table or in DNAs more iconic vertical orientation. For the vertical assembly, you will need the black base and rod that are provided with the 12 Base-Pair DNA Discovery Kit. (Please refer to the printed base and rod assembly directions, on pages 4 and 5.) Correct Pairing for Forming Double Helix Optional Questions & Activities You can use the questions in the yellow boxes on the next three pages to guide your students, if they have difficulty building an accurate DNA double helix. Alternatively, you can use the following questions to prompt discussion after your students have correctly assembled the double helix. If you dont use the questions on the next three pages, skip ahead to the green box entitled, Watson & Crick DNA Papers on page 11. Please also see The Guided Discovery Approach on the website for additional information and ways to use the DNA Discovery Kit. Background Information on DNA is available at 3dmoleculardesigns.com/resources.php. 8

9 Teacher Notes Use your models to build the individual nucleotides (base + deoxyribose + phosphate). What you can discover about how the nucleotides might interact with each other? What general feature of these nucleotide pairs do you see? Remember that the crystallographic data indicated DNA was a helix made up of two strands. Using what we learned previously about the primary structure of DNA, and what youve just discovered about pairs of nucleotides, can you build a helix made up of two strands of DNA? Students who build the correct A - T and G - C base pairs should also discover that they can build the double helix by forming the correct bonds between the 5' phosphates and the 3' hydroxyl groups. They may also find they can form incorrect C - C and G - G base pairs using only two of the three hydrogen bonds, or A - A and T - T base pairs. (Note photo on right.) Incorrect Pairing for Forming Double Helix When the phosphate-deoxyribose is added to the incorrectly paired bases, your students will not be able to add them to correctly paired A - T or G - C base pairs to form a double helix. (Note photo below. Both of the phosphate-sugar groups have a downward orientation. Now note the similar photo on the previous page) Ask your students if they can discover alternative base pairing that will allow them to put two base pairs together. You may want to explain that in a natural environment, nitrogenous bases can form incorrect bonds, but the bonds Incorrect Pairing for Forming Double Helix will be unstable and break apart since they wont be able to bond with correctly paired A - T or G - C base pairs and form the stable double helix structure. After students explore base pairing, have them disassemble the nucleotides and then form a two-nucleotide chain as was done in the first exercise. Then ask the students to use what they learned about base pairing to add two nucleotides to the structure so that they build a helix with two strands as was suggested by the crystallographic data. The students should discover that the only way to build a double helix is by following the A - T, G - C base pairing rule. 9

10 Teacher Notes The 5' phosphate of one strand is opposite the 3' hydroxyl of the opposite strand, and vice versa. Note that the two DNA strands run in the opposite directions. As a result, one strand is oriented in the 5' - 3' direction, while the opposite strand is orientated in the 3' - 5'. The two strands are said to be anti-parallel due to the different directions. Looking at your double helix model of DNA, can you identify the 5' phosphate and 3' hydroxyl of each strand? Where are they in relation to each other on the two strands? The DNA sequence is always read in the 5' - 3' direction. Can you write the sequence of each strand in your DNA model? Check to make sure the sequences are read in the 5' - 3' direction. At this point, it would be useful for your students to assemble all 12 base pairs into the double helix. Lets examine the full DNA model to see if we satisfied all of the experimental predictions. Is it a polymer? Yes. Does it form a double stranded helix with 10 residues per turn as predicted from the x-ray crystallography? Yes. Starting with the 5' phosphate at the top of the model, count down 10 residues. The tenth 5' phosphate will line up directly under the first 5' phosphate. What do you notice about the location of the phosphate molecules and the bases? The phosphate molecules, which are negatively charged, are on the outside where they interact with the aqueous environment. The bases are stacked on top of one another with the planes of the bases nearly perpendicular to the helix axis. How does the base paring in the model explain Chargaffs Rules? While the overall nucleotide composition (percentage of G - C pairs and percentage of A - T pairs) of the DNA of different organisms can vary, the concentration of A always equals the concentration of T, and G is always equal to C. This is a direct consequence of Chargaffs Rules, which state that A is always paired with T, and G is always paired with C. 10

11 Teacher Notes How does this organization of DNA's bases, deoxyriboses and phosphates make DNA a stable molecule? In other words, what forces make this a stable structure? The ribose subunits in the backbone are connected by covalent phosphodiester bonds. The backbone is connected to the nitrogenous bases by covalent bonds. The nucleotide base pairs are formed by hydrogen bonding. A-T base pairs form two hydrogen bonds and are less stable than G-C base pairs, which form three hydrogen bonds. Hydrophobic interactions between the base pairs provide additional stability to the double helix. Reviewing Bonds A covalent bond forms when two atoms share two electrons. A covalent bond is an intramolecular bond within one molecule. Covalent bonds can be either polar (which have partially charged atoms) or non-polar (without charged atoms). A hydrogen bond is an intermolecular force between the two molecules where a positively charged hydrogen atom interacts with a negatively charged fluorine, nitrogen, or oxygen atom in a second molecule. An ionic bond is the complete transfer of an electron between two atoms resulting in one positively and one negatively charged atom. Ionic bonds are intra-molecular bonds within one molecule. Ions are charged atoms that have gained or lost electrons as a result of an ionic bond. Watson and Crick DNA Papers After your students discover the structure of DNA by putting the model together, they should read the classic paper published by Watson and Crick in Nature, April 23, You can download the PDF at 3dmoleculardesigns.com/resources.php. (an annotated version of the paper is included as a teacher resource.) Watson and Crick published a second paper in the next issue of Nature that expanded on the significance of their proposed structure. This paper provides an interesting description of what was known and unknown at that time, and sets the stage for the construction of the Central Dogma of Molecular Biology, which was developed in the following years. One interesting feature of the DNA structure that is addressed in Watson and Cricks second paper concerns the way in which the two strands of DNA wrap around each other. Watson and Crick clearly understood the topological problem this structure presents, even though they did not understand at that time how the cell would deal with it. To help your students better understand that the two strands of DNA are intertwined and to appreciate the problem this intertwining poses, we have included the supplies to create a Mini-Toober model of double-stranded DNA,with the 12 Base-Pair DNA Discovery Kit. Instructions and photos follow on the next two pages. 11

12 Teacher Notes Making a Mini-Toober Model of DNA Place two Mini- Toobers side-by-side, so the red end cap on one is even with the blue end cap on the other. Next, line them up with the first two grooves in the black helix guide. Begin wrapping the Mini-Toobers around the guide following the grooves. You just made a right-handed double helix DNA model. How do you know if your helix is right-handed? Imagine the helix is a spiral staircase. As you walk up, one of your hands rests on the outside rail of the staircase. If it is your right hand, then you are walking up a right-handed helix. Once the Mini-Toobers are completely wound around The structure of DNA is always a the guide, you can remove the Mini-Toobers by twisting right-handed double helix. the guide, as though you are unscrewing it from the Mini-Toobers. Then loosen and separate the coils by gently unwinding and pulling them apart. Carefully position each strand of DNA to show the major and minor grooves (right photo). Then, holding the DNA horizontally by only one strand, demonstrate that the two strands are wrapped around each other(above photo). (The term used to describe this property of DNA is plectonemeic.) Another way to show that that DNA is plectonemeic is to separate the two strands of Mini-Toober DNA, by unwinding one strand from the other (Photo next page top). Separate the Mini-Toobers before class. Once class starts ask one of your students to put the two strands together in the 12

13 Teacher Notes same way that the double stranded DNA fits together. After a few false starts your student will probably try winding one of the strands into the second strand. How do you think the cell separates the two strands of DNA for replication and transcription, when they are wound around each other? Watson and Crick realized the problem intertwined DNA poses for DNA replication and RNA transcription. In their May 1953 paper, published in Nature (see Watson & Crick PDFs online), they wrote,since the two chains in our model are intertwined, it is essential for them to untwist if they are to separate. As they make one complete turn around each other in 34 A, there will be about 150 turns per million molecular weight, so that whatever the precise structure of the chromosome, a considerable amount of uncoiling would be necessary. It is well known from microscopic observation that much coiling and uncoiling occurs during mitosis, and though this is on a much larger scale it probably reflects similar processes on a molecular level. Although it is difficult at the moment to see how these processes occur without everything getting tangled, we do not feel that this objection will be insuperable. We now know that the solution is provided by a family of proteins known as topoisomerases. These enzymes function to unwind or wind double-stranded DNA by: Cleaving a phosphodiester bond in the backbone of one strand of DNA Effectively unwinding the free end one turn around the other DNA strand Re-forming the phosphodiester bond. In this way DNA can be unwound one turn at a time. You can simulate the result but not the mechanism of this localized unwinding by grasping the toober model with both hands spaced about 6 inches apart. Then unwind the double-helix to form the replication bubble shown above. Discussion Opportunity Compare the DNA Discovery Kits plastic model with its Mini-Toober DNA model. What are the advantages and disadvantages of each? Compare them to textbook drawings and illustrations of DNA. (Drawings appear on the next page. Larger images that can be printed on transparency film for overhead use appear on page 20.) 13

14 Three Frequently Asked Questions How do these models compare with the chemical drawings of nucleotides in my textbook? As your students become familiar with DNAs phosphate groups, deoxyribose groups and bases, by handling the models, the 2-D drawings of DNAs chemical structure will be more meaningful. When your students compare the models with the chemical drawings in textbooks, it is important that they understand that most of the hydrogen atoms have been eliminated from the models in order to more clearly reveal the underlying structure. A direct comparison of the physical models with a typical chemical drawings of the nucleotide structures is provided below. G C A T 14

15 Three Frequently Asked Questions How does the model show that the two strands of DNA are anti-parallel? One powerful feature of this model is that it clearly demonstrates that the two strands of DNA are running in opposite directions. Look at the photo shown below, and focus on the red oxygen atom found in the two deoxyribose groups. Notice how the oxygen of the deoxyribose on the left is below the plane of the base pair, while the oxygen of the deoxyribose on the right is above the plane. This is a clear indication that the polarity of the nucleotides in the two strands are opposite each other Now focus your students attention on the phosphate groups from each nucleotide. Again, one of these phosphates will be below the plane of the base pair while the other will be above the plane. And since the phosphate group is attached to the 5carbon of the deoxyribose group, the DNA chain on the right of the double helix shown in the photo above is said to run 5 to 3 from the top of the photo to the bottom while to other strand is running 5 to 3, from the bottom to the top. 15

16 Three Frequently Asked Questions Can incorrect base pairs be formed with the model pieces? Yes, non-standard base pairs (other than the A - T and G - C that form the double helix) can be formed by this model just as these base pairs can form in solution with real nucleotides. Four of these non-standard base pairs are shown below. However, these non-standard base pairs are not compatible with double helical DNA, for two reasons. Base pairs formed with two purines or two pyrimidines will have a different diameter than standard A - T and G - C base pairs that consist of one purine paired with one pyrimidine. Therefore, the non-standard base pairs shown below cannot be assembled into a double helical model with a uniform diameter. Encourage your students to discover these non-standard base pairs -- and then determine for themselves why these base pairs are not consistent with the model proposed by Watson and Crick. For the non-standard hydrogen bonded base pairs to form, the polarity of the two strands of DNA must be parallel, not anti-parallel. Therefore, notwithstanding the problem with the diameter of these non-standard base pairs (see above paragraph), it is not possible to accommodate these parallel base pairs in the Watson-Crick model of DNA. 16

17 ...where molecules become real TM The Discovery of the Structure of DNA On April 25, 1953, a one-page paper entitled, A Structure for Deoxyribonucleic Acid, appeared in the British journal, Nature. The authors of this paper were James Watson, a young American post-doctoral candidate who had recently received a Ph.D. from the University of Illinois, and Francis Crick, a physicist who was completing his doctoral dissertation at Cambridge University, England. The paper began; "We wish to suggest a structure for the salt of deoxyribose nucleic acid (D. N. A.). This structure has novel features which are of considerable biological interest." This initial description of the structure of DNA marked a major milestone in the development of molecular biology. In addition to reporting the correct structure of DNA, the paper also contained their classic understatement in scientific literature: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Their paper serves as an excellent example of what has become a recurring theme in the molecular biosciences Forms Follows Function. That is, the structure of a macromolecule often explains the macromolecules function (how the macromolecule) works. Watson and Crick's achievement is notable in several ways, including the fact that they determined the structure of DNA without performing a single experiment. They used the information from numerous other scientists who were investigating various properties of DNA. Modeling was the major approach Watson and Crick used. Using paper cut-outs of the shapes of the four nitrogenous bases (A,T, G and C), they were able to combine all of the different facts that had accumulated to that date into a plausible model for the structure of DNA....The structure has two helical chains coiled around the same axis (see diagram). We have made the usual chemical assumptions, namely, that each chain consists of phosphate diester groups joining B-D-deoxyribofuranose residues with 3',5' linkages. The two chains (but not their bases) are related by a dyad perpendicular to the fibre axis. Both chains follow right-handed helices, but owing to the dyad the sequences of the atoms in the two chains run in opposite direction. Watson, J.D. and Crick, F.H.C., Nature, 171, (1953) (Page 1 of Student Handout) 17

18 Student Handout The DNA Student Challenge Your challenge today is to see if you can discover the correct structure of double-stranded DNA, just as Watson and Crick did over 50 years ago. Your model should satisfy all of the pieces of experimental information that was known in 1953, as noted in the blue box below. Rather than using paper cut-outs to represent the DNA bases, you will use plastic models of the four deoxyribonucleotides whose 3D structures are based on known atomic coordinates of the B-form DNA. In these nucleotide models, magnets are used to represent both: the phosphodiester bonds that link the nucleotide units together into a long, linear polymer the hydrogen bonds that bond one base to another. Information Available to Watson and Crick in 1953 DNA is a Polymer: Previous studies identified DNA as the genetic material of cells, and that DNA was a polymer consisting of three components: A nitrogenous base A pentose (5-carbon) sugar called deoxyribose A phosphate group. Moreover, experiments suggested that the DNA molecule was unbelievably large, with molecular weights ranging from 25 x 10 6 to 3 x 10 9 daltons. (Since each nucleotide has a mass of 330 daltons, DNA molecules were believed to be composed of between 76,000 and 9,000,000 nucleotides.) DNA is more dense than protein. At a density of 1.6 gm/cm 3, DNA was known to be more dense than protein (1.3gm/cm 3 ). This suggested that DNA was a densely packed structure. Chargaff's Rules: In 1947, Erwin Chargaff demonstrated that while the four nucleotides were not present in equal amounts in the DNA from different organisms, the amount of adenine was the same as thymine, and the amount of guanine was the same as cytosine. This became known as Chargaff's Rules: The proportion of A always equals that of T, and the proportion of G always equals that of C. Thus, A = T and G = C. X-ray Crystallography Data: In the laboratory of Maurice Wilkins, Rosalind Franklin used X-ray diffraction to analyze fibers of DNA. The pattern of spots on the X-ray diffraction pattern suggested that: Phosphate was on the outside, nitrogenous bases were on the inside. DNA was a double helix, made up of two strands. The two strands of DNA run in opposite directions (anti-parallel). There are 10 base pairs per turn of the double helix. (Page 2 of Student Handout) 18

19 Background information for students Phosphates Deoxyribose Nitrogenous Bases Each group of students should have physical models of the four nucleotides, separated into their component parts. These include: Phosphate group which is negatively charged Deoxyribose group which is a cyclic ring structure Four nitrogenous bases (A, G, C and T) Each component of the nucleotides is color coded according to atom type, following the standard CPK coloring scheme: Oxygen is RED Nitrogen is BLUE Phosphorus is YELLOW Carbon is GRAY Hydrogen is WHITE (Page 3 of Student Handout) 19

20 G C A T Transparency Template of Comparison of Models to Textbook Drawings of Nucleotides. See page

21 Information Available to Watson and Crick in 1953 DNA is the genetic information of cells DNA is a Polymer A nitrogenous base A pentose (5-carbon) sugar called deoxyribose A phosphate group. DNA molecule is unbelievably large DNA is more dense than protein Chargaffs Rules The proportion of A always equals that of T The proportion of G always equals that of C A = T and G = C X-ray Crystallography Data The phosphate is on the outside; nitrogenous base is on the inside. DNA is a double helix, made up of two strands. The two strands of DNA run in opposite directions (anti-parallel). There are 10 base pairs per turn of the double helix. Transparency Template of Information Available to Watson & Crick. See pages 6 and 18. 3D Molecular Designs Copyright -- 21