Manipulation and Analysis of DNA

      The Polymerase Chain Reaction: Paper PCR

      Objectives

      After this lesson, students should be able to
      1. Describe the steps in the PCR and explain how these steps can generate multiple copies of a specific DNA fragment;
      2. Describe how PCR can be used in disease diagnosis.

      Materials

      • Strips of light- and dark-colored paper (at least eight of each per student group)
      • Removable tape (one roll per student group)
      • Scissors (one pair per group)
      • At least eight light and eight dark primers per student group (Appendix)
      • One "double-stranded" parental DNA molecule per group (Appendix)
      • One "sample 1" and "sample 2" (Appendix) page per group
      • One copy of student questions per student

      Teaching Resource

      Carolina Biological Supply Co. sells an instructional video (catalog no. 21-2734) in which the model presented in this activity is used to explain how PCR works. The video also demonstrates a PCR laboratory activity that does not require a thermal cycler (see PCR in the Classroom, below).

      Preparation

      Cut the light and dark paper strips to about the width of the primer and template models.

      If students do not have manuals, photocopy the page with the parental DNA sequence and the primers as many times as needed for your class. For the part of the activity that illustrates the use of PCR in disease diagnosis, make enough photocopies of the "Sample" pages for your class (or make transparencies).

      Procedure

      PCR

      Refer to Fig. 18.3 through 18.8.

      Each student group has a parental DNA molecule, primers, and strips of colored paper that will represent newly synthesized DNA (these strips are somewhat analogous to the free nucleotides in the solution). The two parental strands should be taped together to form a double-stranded molecule. There is no representative of the DNA polymerase in this model; the DNA "synthesis" will be performed by the students (Fig. 18.3).

      Step 1. Denaturation

      This is the 95°C step. Students should denature their double-stranded parental DNA into two single strands by removing the tape holding the strands together.

      continued...

      Step 2. Hybridization

      The temperature is reduced, and hybridization can occur. Because there are so many primers in solution, they will hybridize to the parental DNA before the two parental strands can find each other (ideally, students should have many more than eight primers of each color on their desks). Students should "hybridize" the primers to the long DNA strands by matching the primer sequences to their complementary sequences and taping the primer in place. Be sure that the students hybridize the primers in the correct orientation: if the parental strand is oriented with the 5' end on the left, then the 3' end of the primer should be on the left. It doesn't matter if the letters are upside down. Real DNA strands are twisted into a helix, so there is no upside down. Because of the way the paper primers are designed, a dark primer should always hybridize to a light DNA strand (Fig. 18.4).

      Step 3. DNA Synthesis

      Students will "synthesize" DNA by taping one end of a strip of light-colored paper to the 3' end of the light primer that is hybridized to the dark single strand. The light-colored strip of paper (the new DNA strand) should be extended to the end of the parental DNA "template," and then any excess paper should be cut off. The new light-colored DNA strand should be taped to its complementary dark strand to represent the new double-stranded DNA molecule. Finally, students should write the correct DNA sequence on the new strand. Go through the same procedure with the dark primer that is hybridized to the light-colored strand, and synthesize a new dark strand.

      After one round of synthesis, there will be two double-stranded DNA molecules (one end of each will be uneven; Fig. 18.5).

      Now, repeat steps 1 through 3 with the two DNA molecules. Note: The primers that started the synthesis of strands in the previous round are now part of the new DNA strands. They must be used as part of the new templates, too 18.6). The products will be four double-stranded molecules (Fig. 18.7). Notice how the length of the new strands is changing.

      Repeat steps 1 through 3. This repetition produces eight double-stranded molecules, of which two stretch only from one primer sequence to the other (Fig. 18.8); note the two products with even ends stretching between the primer hybridization sites).

      Ask your students to predict the products of another round of synthesis. Predicted products can be drawn on paper or the blackboard or done with paper materials. (If you have students perform another round of paper synthesis, they will need additional light and dark paper strips [eight of each] as well as additional light and dark primers [eight of each].) There will be 16 molecules, and 8 of them will be the short species. Yet another round will give 32 molecules, of which 22 will be the short species. A sixth round of synthesis yields 64 helices, of which 52 are short. The bottom line is that the number of molecules with primer sequences at their ends increases dramatically, forming the vast majority of the products.

      It is important for students to see that the products of PCR are almost all identical and that they are double-stranded DNA segments that begin and end with the two primer sequences.

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