Manipulation and Analysis of DNA

      The Polymerase Chain Reaction: Paper PCR

      DNA analysis with PCR

      Hand out the sample 1 and 2 DNA and primer sequences to your students if students do not have manuals. Have them cut out the primers. Tell the students that the two sample sheets represent DNA prepared from two different sources. The students are now laboratory technicians, and they will perform PCR on both samples, using the primers provided (they are to assume that they have large quantities of the primers).

      Have students predict the DNA sequences of the major products from the two separate reactions.

      The product from sample 1 (primer sequences underlined) is

      TTCCAGCCAGAGTCT
      CGGAACTAGCCTTATG

      AAGGTCGGTCTCAGAG
      CCTTGATCGGAATAC

      There is no product from sample 2:
      primers do not hybridize.

      The students will notice that "something is wrong" with sample 2 and that the primers do not hybridize anywhere. Get them to talk about this. Ask them what they would see if they simply performed the reactions with the two samples, loaded the results into a gel, electrophoresed, and stained. (Answer: A product band in the sample 1 lane but none in the sample 2 lane.) Ask them if they think it would be possible to use PCR to determine whether a specific microorganism was present in a sample or whether a specific gene was present in an organism.

      Explain to the class that PCR can be used to detect the presence of disease-causing microorganisms in medical specimens. DNA is prepared from a sample taken from the patient (blood, tissue, sputum, etc.), and PCR is performed with primers that hybridize only to the DNA of the microorganism of interest. Laboratory workers can then tell if the microorganism was present in the sample by whether or not any DNA product is produced. This kind of test can be used in disease diagnosis. continued...

      Have students answer the questions (in class or for homework).

      Answers to Student Questions

      1. She would have to know enough of the DNA sequence of the virus to design primers that would hybridize to it. She would have to test to make sure that those primers did not hybridize to human DNA or to samples prepared from healthy patients (which might contain small amounts of DNA from other microorganisms). She could use computerized DNA sequence comparisons for some of her primer designing (to make sure the primer sequence was not present in any known organisms other than virus X).

      2. Make the primers long. The longer the primer, the less likely that it will accidentally hybridize to other DNA molecules, since the odds of a given base sequence occurring randomly are 1 in 4n, where n is the number of bases in the sequence (see chapter 16).

      3. Answer: 2n

      4. Answer: 2n

      5. No. With only one primer, you would be limited to synthesizing one strand over and over and could never synthesize the complementary strand. After one round, you would have one new single strand. For the second round, you would still have only one template for the primer, so you could generate only one more new single strand, and so on.

      Selected Readings

      Mullis, K.B. 1990. The unusual origin of the polymerase chain reaction. Scientific American 262(4):56-65.

      Paabo, S. 1993. Ancient DNA. Scientific American 269(5): 86-92.

      PCR in the Classroom

      If you do not have a thermal cycler

      Carolina Biological Supply Co. sells kits (catalog no. 21-1220, 21-1222, and 21-1224) for classroom PCR that you can use even if you do not have a thermal cycler. They require two water baths, one boiling and one set at 55°C. Teacher and student directions are included. You must supply electrophoresis equipment to separate the products. These demonstration kits have been specifically designed to work with the less than ideal conditions of two water baths. The water baths will not give detectable products with commercial kits that require a thermal cycler. Carolina Biological Supply Co. also sells a videotape (catalog no. 21-2734) in which this activity is demonstrated.

      If you have a thermal cycler

      The experiment kits listed above, which were designed for teaching, can be used with a thermal cycler.

      You can also order research-grade kits from Perkin-Elmer Corp., 761 Main Ave., Norwalk, CT 06854 (800-762-4002). Call and ask for a catalog.

      If you go on to the lesson about DNA typing (chapter 24), you can order and perform PCR-based DNA typing with an educational kit from Carolina Biological Supply Co. Their kit "Human DNA Fingerprinting by Polymerase Chain Reaction" (catalog no. 21-1226) provides reagents needed to amplify a VNTR region on chromosome 1. You must supply electrophoresis equipment to separate the PCR products.

      Page 3

      Page 1 | Page 2 | Page 3 | Page 4
       
      Feedback   About AE   Discussions   Copyright © Info   Privacy Policy  
      Sitemap  Email this Link   Contact   Access Excellence Home