Lesson 7: Lo-Tech PCR

Introduction:

This simulation and manual PCR demonstration is designed for classrooms without a thermocycler and those that do not have enough time to do a full manual PCR. It is designed to give students the concepts and vocabulary as well as give them a visual representation of the process.

Learning outcomes:

Students will be able to describe the process and product involved in a PCR reaction.

Curriculum alignment:

Biology Goal 3.04 – applications of biotechnology

Classroom time required:

60 minutes: Reading up to “Procedure” – 10 minutes, explanation of procedure -10 minutes, following procedure – 20 minutes, answering questions – 20 minutes

Materials needed:

Per each student: Handout of “Lo-Tech PCR” activity

Per group of 2 students: Original DNA template (copied on blue paper, not cut out), forward primers (copied on yellow paper, cut out), reverse primers (copied on green paper, cut out), Ziploc bag labeled “Free Nucleotides” containing cut out nucleotides (A’s, T’s, G’s, and C’s) that have been copied on white paper, 1 pair of scissors, 1 roll of clear tape

Per class for demo: 3 water baths, 3 thermometers, bucket of ice, 1 microcentrifuge tube filled with water (students will be told this contains all necessary reactants for PCR; however, this is not necessary for this demo as it will not be used to run a gel electrophoresis), Styrofoam holder for centrifuge tube, test tube, deionized water, cotton swab, hot plate or microwave with beaker of water to hold test tube, glass stirring rod, 2 pair of student goggles

Technology resources:

Overhead projector

Pre-activities:

Make a copy of each of the template sheets onto overhead film and cut out as it will be for the student set. You will be walking them through the first cycle. Make copies of activity for each student. Also, prepare copies of “Original DNA” template, forward and reverse primers and all nucleotides. Leave the “Original DNA” template intact to give to each group of 2, cut out the nucleotides individually and place them in the Ziploc and label. Finally, cut out the primers label the back “F” for forward and “R” for reverse. Paper clip these separately to make them easier to hand out. Set up water baths, one at 94oC, one at 48oC, and one at 72oC prior to class. Set up station for demo group using materials listed above. Be sure to have water in microcentrifuge tube prior to class. Students should already be familiar with DNA synthesis during the S phase of cell division as well as the structure and function of DNA.

Activities:

1. Review with students the parts of a nucleotide that make up DNA and what holds nitrogen bases together. Also, ask students the function of DNA for review. Ask students how they have seen DNA used to solve a problem (perhaps on CSI). Finally, ask students what they think would happen of only a tiny amount of DNA was available from an organism for use. Lead them to the response that copies of DNA would need to be made.
2. Hand out copies of Lo-Tech PCR activity to each student. Have students get into groups of 2. Hand out copies of the “Original DNA” template, forward ad reverse primer sets and “Free Nucleotide” Ziplocs.
3. Have a student read 1st paragraph for background information to the entire class. Then, have students read up to the “Procedure” individually.
4. Select one group of students to be the “demonstration group.” They will perform the demonstration for the class under your direction. Have these students put on goggles and check their attire and hair to meet lab safety requirements.
5. Explain steps 1-4 of the procedure to students, pausing after each step to allow students to complete the task. At the end of step 4, make sure that all groups have 2 identical pieces of double-stranded DNA.
6. Instruct students to perform the procedure 2 more times and then answer the follow up questions. Be sure to rotate through the groups to help students perform the steps in order and review new vocabulary in reference to the follow up questions.
7. Students can place their 8 complete DNA strands in the Ziploc bag to turn in with the activity. Have the demonstration group turn off water baths, throw away cotton swabs, remove Styrofoam and reaction tube from water bath, and rinse out the test tube and stirring rod upon completion.

Assessment:

Check responses to follow up questions for accuracy as well as the DNA pieces to make sure all 8 are identical or the original. Verbally assess students when papers are turned in to see that they understand what PCR means, what they steps are, what it produces, and what it can be used for.

Modifications:

Students with learning difficulties would benefit from having the section under the background paragraph read to them. This could be accomplished by having one person in each group of 2 read aloud or you could read and/or explain this section to the entire class. Also, show students how to calculate 2x if they are uncertain how to find this value on the calculator.

Supplemental information:

Extensions:

1. 1. Have students watch video on (website to be built) in the steps involved when using an actual thermocycler. Discuss with the class that this machine goes through the same process that the demonstration group performed, but it does so with just the touch of a few buttons.
2. 2. Show students the “PCR song” from Bio-rad: http://www.cnpg.com/video/flatfiles/539/ The song is done in a style-similar to “We are the World” and is meant to be funny. Have students then write their own song to explain the process and/or benefits of PCR. It is best to let them choose a tune first and then make up words to that tune.

Critical vocabulary:

• Enzyme: proteins that are produced by living cells and catalyze specific biochemical reactions at specific temperatures
• Complementary nucleotides: In DNA, adenine correctly pairs with thymine and guanine correctly pairs with cytosine. Therefore the complement is the nitrogen base that correctly pairs with the base that is given.
• Denature: to modify the molecular structure of (as a protein or DNA) especially by heat, acid, alkali, or ultraviolet radiation so as to destroy or diminish some of the original properties and especially the specific biological activity.
• Anneal: to be capable of combining with complementary nucleic acid by a process of heating and cooling.
• Elongate: addition of free nucleotides to an exposed single strand of DNA during replication.
• Thermocycle: alternation of various temperatures to complete different reactions such as those in the PCR process
• Primer: A primer is a strand of nucleic acid that serves as a starting point for DNA replication.
• Free nucleotides: Sugar, phosphate, and nitrogen base complexes that are unattached to a DNA or RNA strand and are available for base pairing.

Websites and Resources:

• Genetics Learning Center: http://www.cnpg.com/video/flatfiles/539/
• Dolan DNA Learning Center: http://www.dnalc.org/ddnalc/resources/pcr.html
• Washington University Genome Sequencing Center: (This lesson was based partially off of this sites lesson) http://www.nslc.wustl.edu/elgin/genomics/gsc/PaperPCR.pdf
• PCR Time Warp (another inspiration for this lesson): http://www.woodrow.org/teachers/esi/2002/Biology/Projects/p3/pcrtimewarp...

Comments:

Once a “gene of interest” is located from studying phenotypes of offspring of an organism, that gene may be amplified for further study. PCR is the process of amplification. The next step would be to run the DNA through a gel electrophoresis and compare it to other known genes. These are initial steps for being able to use these genes for biotechnology products and processes.