Teachers Guide: Polymerase Chain Reaction (PCR)

Several recent studies suggest that a small piece of DNA, called an Alu element, found its way into the human Tissue Plasminogen Activator gene (TPA) less than one million years ago. In a given group of individuals, some carry this 300 base pair insertion while others do not. During this laboratory investigation, student's will have an opportunity to extract their own DNA from a cell sample taken from the lining of their mouth and to use a powerful molecular biology technique, called the Polymerase Chain Reaction, to amplify a region of the TPA 25 gene to determine if they carry this Alu insertion. The region targeted for amplification is located in an intron (noncoding region) of the Tissue Plasminogen Activator gene, mercifully abbreviated TPA 25.

The polymerase chain reaction, PCR, is a molecular biology technique that was developed by Dr. Kary Mullis during the early 1980's. The technique uses some elegant chemistry and precise thermal cycling of the reactants to target a specific piece of DNA and then to use that piece as a template to produce billions of copies. From a single DNA fragment, PCR can produce over one billion copies of this fragment in three hours. This elegant piece of science proved so significant that Mullis was awarded the Nobel Prize for Chemistry in 1993. Introduced to the public in 1985, this procedure proved to be so significant that by 1993, it was used and cited in over 7,000 scientific publications. Today, PCR is considered to be a standard protocol in molecular biology and thousands of scientific papers each year.

Purpose

This laboratory protocol will allow students to demonstrate the phenomenon of polymerase chain reaction, the amplification of a small piece of DNA so that it can be visualized using gel electrophoresis.

 

PCR Learner Outcomes

The following cognitive objectives should be addressed. The student will be able to:

1. Define the vocabulary related to PCR: DNA, Taq polymerase, primer, denature, anneal, elongate, target sequence.
2. Describe the work of Kary Mullis as it relates to PCR.
3. State why PCR is used to identify many bacteria and viruses.
4. Relate genetic constitution of an organism with its physical attributes: describe the relationship between genotype and phenotype.
5. Apply the technology used for PCR to current work in forensics and genetic testing.
6. Discuss the moral and ethical issues of determining a person's genotype.

The following laboratory objectives should be addressed. The student will be able to:

1. Pipette reagents.
2. Wash a pelletized cell sample.
3. Load and run a gel for electrophoresis.
4. Compare experimental data with accepted values.