The polymerase chain reaction (PCR) has become a mainstay of modern molecular biology since its discovery in the 1980s. Discover the principles of this DNA amplification process during this activity.
This activity assumes a knowledge of basic DNA structure and chemistry. If basic DNA structure is not known, please consult basic text materials before starting this activity.
The polymerase chain reaction (PCR) is a very important research tool for the molecular biologist and has been used extensively in forensic settings since its discovery. The PCR’s ability to amplify (make billions of copies of a segment of nucleic acid) is based upon the chemical properties of DNA itself. PCR is the equivalent of DNA photocopying. A single molecule can be duplicated into billions of molecules in just a few hours. Producing a large predictable sample from a single strand of DNA can be extremely useful in genetic research and in criminal investigations. The discovery of the PCR process will surely be noted as one of the great discoveries in molecular biology in the 20th century.
The structure of DNA and its ability to replicate itself in a semiconservative manner is the basis behind PCR. Small segments of genetic information coded in the DNA molecule can be amplified with PCR to make a large quantity of identifiable and analyzable material. When DNA is heated to 94 °C, the hydrogen bonds between the base pairs break and DNA becomes single-stranded. The two strands can come back together (reanneal) when the temperature is lowered to 55 °C or less. DNA polymerase is the key enzyme responsible for creating a complementary copy of the original DNA at a specific region.
The basic PCR procedure is completed in three major repeating steps (see Figure 1):
- The DNA to be copied is extracted from the cells. This is followed by heating the DNA to about 94 °C to “separate” it into single strands.
- Next, the strands are cooled to about 50 °C so that primers can anneal to the single strands of DNA. The primers bracket the “target” strand of DNA and provide the specificity for the PCR process.
- In the third step, the strands are heated to 72 °C and the DNA polymerase extends the primers by adding the nucleotides needed to make a complementary strand of DNA that includes the target.
Steps 1–3 are then repeated 30–40 times and the number of copies increases exponentially. Theoretically, one cell can provide a billion copies of the target in 30 cycles.
The target may be a gene or a segment of DNA that interests the investigator performing the PCR procedure. The target is a unique sequence of nucleotides from 100–1,000 base pairs long. A target of 200–500 base pairs is considered to be an optimal size target. Most of the sequences in the target must already be known in order to choose unique primers.
Primers are short, single-stranded, oligonucleotides that bracket the target. Primers are synthesized using an automatic procedure that is relatively inexpensive or they can be purchased from a supplier that specializes in customizing primers. Two primers are used in PCR. One primer is a copy of a short section of the coding strand of DNA at the 5′ end of the target and the other is a copy of the noncoding strand at the opposite 5′ end of the target. Primers are usually 20–30 nucleotides long and must not be complementary to each other. They also must be unique and only anneal to the target. Because the primers are short and added in excess when the mixture is cooled during the PCR procedure, they anneal to the target DNA before the long strands of target can come back together. The primers provide a starting point for the DNA polymerase enzyme to synthesize a second strand complementary to the first.
Most enzymes are denatured and destroyed at high temperatures. The DNA polymerase most commonly used in PCR (thermus aquatus or Taq) is unique in that it can withstand high temperatures. It was originally isolated from a bacteria strain that lives and thrives in hot springs. The isolation of this heat-resistant Taq polymerase was a critical step in the development of the PCR process.
The materials required in the PCR amplification chamber are a brew of key ingredients necessary for DNA replication to occur. The materials include an excess of four deoxynucleotide triphosphates (adenine, thymine, cytosine and guanine), DNA polymerase, an excess of primers, buffers and magnesium chloride.
The thermal cycler itself is a programmable, microprocessor-regulated, heating and cooling block. The instrument can be programmed for the temperatures and time required for each step of the PCR procedure. In a typical procedure, the specimen to be amplified and the other ingredients are placed in a tiny, thin-walled test tube. The tube is placed into the well of the thermal cycler, heated to 94 °C for one minute for dissociation, cooled rapidly to about 50 °C for one minute to allow the primers to anneal and then reheated to 72 °C for one minute for the polymerase to extend the primers. Forty cycles of this duration can be completed in two hours. Different procedures may have different optimal times and temperatures depending on the length of the target, the length of the primers and the predominant bases in the DNA.
In this activity, the PCR procedure will be simulated and the nature of the amplified pieces will be analyzed.
Pencils, three colors
Primer Cutouts, red
Primer Extender Cutouts (long and short), yellow
- Locate a large, flat, clutter-free workspace. A cleared lab table or countertop is ideal.
- Obtain two complementary DNA strands. Cut out the primers and primer extenders from the printed sheets.
- Place the DNA strands in the center of your work area with base pairs aligned properly in a 3′–5′ orientation.
- Locate two primers that will anneal to the DNA strands. Remember to follow the 3′–5′ orientation.
- Place multiple copies of these two primers randomly around the DNA strands.
- Simulate the initial heating of the mixture to 94 °C. (Pull the DNA into two single strands. Move the strands far apart.)
- Simulate cooling the mixture to 50 °C. (Find a location on each single-stranded DNA to anneal one primer.)
- Simulate the heating of the mixture to 72 °C. (Extend the primers along the length of each DNA strand. Find a blank strip that extends to the end of the DNA strands. Once again note the proper 3′–5′ orientation.)
- Use a small strip of cellophane tape to tape the primers and the extenders together into one long piece.
- Starting at the primer end, write in the appropriate complementary base symbols (A, T, C, G) that represent the replication process of each DNA strand. Do this for both strands.
- Now, “heat” the mixture to 94 °C again. (Separate the two DNA strands into four single strands.)
- Complete cycle 2. (Use additional primers and extenders to create four double-stranded DNA molecules. Complete the base pair sequences appropriately on all strands.)
- Complete a cycle 3 resulting in eight double-stranded molecules. Follow the same procedures, taking care to keep track of all segments.
- When all eight molecules are completed, record the data on the PCR Worksheet and then answer the questions.
- Discuss the PCR process and further applications as directed by your instructor.