Manipulation and Analysis of DNA

The Polymerase Chain Reaction: Student Activity

Introduction

In 1985, a new technique that changed the whole picture was introduced. This technique essentially allows a scientist to generate an unlimited number of copies of a specific DNA fragment. It was invented by biotechnology industry scientist Kary Mullis, who had the initial inspiration one night in 1983 as he was driving and thinking about a technical problem he faced at work.

The essence of Mullis's idea was this. If you could set up a test tube reaction in which DNA polymerase duplicated a single template DNA molecule into 2 molecules, then duplicated those into 4, then duplicated those into 8, then 16, then 32, etc., you would soon have a virtually infinite number of copies of the original molecule. Each round of DNA synthesis would yield twice as many molecules as the previous round: a chain reaction producing specific pieces of DNA. Mullis's new technique was called the polymerase chain reaction, or PCR.

Of course, Mullis did more than just realize that DNA polymerase can copy one DNA helix into two. You already knew that, too. What he did was figure out how to generate a chain reaction in a test tube and how to get the reaction to copy the DNA segment of the scientist's choosing.

Mullis's approach relies on the characteristics of DNA polymerase enzymes and on the process of hybridization. Recall that DNA polymerases must have a primer base paired to a template DNA strand so that they can synthesize the complement to the template strand. Also remember that hybridization is the spontaneous formation of base pairs between two complementary single strands: you can separate the two strands of a helix by heating, but if you then allow the mixture to cool, the base pairs between the strands will re-form.

Now, how did Mullis use DNA polymerase and hybridization to get a chain reaction of DNA synthesis? Refer to the diagram in Fig. 18.1 during this explanation.

First, you decide what DNA segment you wish to duplicate (scientists say amplify instead of duplicate, because they are making so many copies). Then you synthesize two short single-stranded DNA molecules that are complementary to the very ends of the segment. These two short molecules must have specific characteristics. Look at the first panel in Fig. 18.1 under round 1. It shows a double-stranded parental DNA molecule with two copies each of the two short, single-stranded DNAs. Each of the single-stranded molecules is complementary to only one strand of the parental DNA, and each one is complementary to only one end of the segment. Furthermore, if you imagine these short molecules base paired to the complementary regions in the duplex, their 3' ends would point toward each other. These short, single-stranded molecules are the primers.

To begin the chain reaction, a large number of primers are mixed with the template molecule in a test tube containing buffer and many deoxynucleoside triphosphates. (What are they for?) This mixture is heated almost to boiling, so that the two strands of the parental molecule denature (Denaturation in Fig. 18.1).

Next, the mixture is allowed to cool. Ordinarily, the two strands of the parental DNA molecule would eventually line up and re-form their base pairs. However, there are so many molecules of primers in the mixture that the short primers will find their complementary sites on the parental strands before the two parental strands can line up correctly for base pairing. So a primer molecule hybridizes to each of the parental strands (Hybridization in Fig. 18.1).

Now DNA polymerase enzyme is added. The primers hybridized to the single-stranded parental molecules meet the requirements of the enzyme for DNA synthesis. DNA polymerase begins adding the correct deoxyribonucleotides to the 3' ends of the primers, forming new complementary strands (DNA synthesis in Fig. 18.1).

After a short time, the mixture is heated up again. Now the two new double-stranded molecules denature, leaving four single strands (Denaturation, round 2). The mixture is cooled, and the abundant primer molecules hybridize to the single-stranded molecules (Hybridization, round 2). DNA polymerase is added again, and new deoxynucleotides are added to the 3' ends of the hybridized primers, yielding four double-stranded molecules (DNA synthesis, round 2). Notice that two of the newly synthesized strands begin and end at the primer hybridization sites.

This process of denaturation, hybridization, and DNA synthesis is repeated over and over, often 25 to 30 times, yielding huge numbers of molecules. The overwhelming majority of the newly synthesized molecules reach exactly from one primer hybridization site to the other. So by choosing the primers, a scientist controls which segment of the parental molecule is amplified. PCR is now used routinely for many different purposes: to amplify a specific fragment of DNA for cloning, to generate a DNA fingerprint from a minute sample, and even to diagnose diseases.

One technical improvement to the process outlined previously has made performing PCR even easier. Did you notice that we added DNA polymerase before each DNA synthesis step? That is because PCR was first carried out with Escherichia coli DNA polymerase, which is rendered inactive at the high denaturation temperature. So more enzyme had to be added for each round of synthesis. However, some organisms inhabit the very hot waters of hot springs and thermal ocean vents. The DNA polymerase enzymes from these organisms are not inactivated by the temperatures required for DNA denaturation. Now PCR is carried out with heat-resistant DNA polymerase, so the enzyme needs to be added only once at the beginning of the reaction cycles.

When PCR was first developed, scientists preset three water baths to the temperatures required for denaturation, hybridization, and DNA synthesis. They performed PCR by simply moving the reaction tube from one water bath to another. It wasn't long before enterprising biotechnology companies manufactured incubators that rapidly cycled between the desired temperatures, eliminating the need for manually moving tubes. These thermal cyclers have further simplified the performance of PCR. PCR can now be carried out by mixing parental DNA, primers, buffer, deoxynucleotides, and heat-resistant polymerase in a reaction tube; placing the tube in the thermal cycler; programming the thermal cycler to the desired time and temperature specifications; and waiting for the cycles to finish. It usually takes a few hours for many cycles of amplification. Figure 18.2 shows a scientist placing a PCR reaction tube into a thermal cycler in his laboratory.

When we (the authors writing this material) and other scientists first learned about PCR, the idea made perfect sense. However, we did not completely get it until we had worked through several reaction cycles ourselves, drawing the parental DNA molecules, the primers, the products, etc. Because we really learned PCR by working through it, we have provided a paper simulation for you to do the same thing. The template and primers are included in this chapter. Your instructor has directions, though you may be able to figure it out yourself. We have also included materials to simulate a PCR-based diagnostic test.

If you do not do the paper simulation, we highly recommend that you get some clean paper and draw through four rounds of PCR yourself. Figure 18.1 should get you started. We like using different colors of pens to keep track of the parental DNA, the primers, and the newly synthesized molecules (remember, primers will form part of the new molecules).

Here are some questions to think about.

Questions

1. What would a scientist have to know before she could design a PCR-based diagnostic test for virus X?

2. How could a scientist help ensure that her primers will hybridize only to the DNA she wishes to detect?

3. Write an expression that predicts the number of product molecules generated from a single double-stranded DNA molecule after n rounds of synthesis.

4. Predict the number of product DNA strands that are not the short primer to primer strands, that would be generated from one double-stranded DNA molecule after n rounds of synthesis.

5. Could you amplify DNA given only one primer? What would the products be after one round? Two rounds? Four rounds?