Manipulation and Analysis of DNA
The Polymerase Chain Reaction: Paper PCR
About This Activity
The polymerase chain reaction (PCR) has become a key tool in molecular biology research and biotechnology applications. PCR is not conceptually difficult if students have sufficient preparation (see below). However, most people, whether school-age students or adult scientists, will not really grasp what is going on until they have gone through all the steps themselves, either by drawing pictures or by manipulating models. In this lesson, students will use paper models to simulate the steps of the PCR. The model exercise demonstrates how DNA polymerase can be used to make multiple copies of a specific DNA fragment and shows how the technique can be used to detect a specific DNA molecule (such as the chromosome of a disease-causing microorganism) in a sample. Students should have performed both activities in DNA Replication and have been introduced to hybridization before performing this activity.Figures 18.1 and 18.2 are shown in the Student Activity pages.
Class periods required: 1-2
Introduction
The PCR has become very popular with both researchers and clinicians. This clever technique allows many copies of a specific DNA segment to be produced from a single copy and is extremely useful as a detection method and a cloning tool. For example, PCR can be used to identify viruses, to analyze scarce DNA (such as from small bloodstains), or even to probe antique DNA from museum specimens.PCR is a simple technique that combines in vitro DNA synthesis by DNA polymerase and hybridization. It requires a solution containing a starting sample, two primers (many molecules of each), a DNA polymerase enzyme, and free nucleotides. The primers are short single-stranded pieces of DNA, typically 16 to 20 bases long. One primer of the pair hybridizes to each of the two strands of the sample DNA at either end of the segment to be amplified. The primers are mixed in great excess with the sample DNA. To start the reaction, the double-stranded sample DNA in the mixture is denatured into single strands by heating (usually to 95°C). The mixture is then cooled so that hybridization of complementary strands can occur. Because the primers are in such great excess, one primer molecule will anneal to each single strand before the strands of sample DNA can find one another(Fig. 18.1).
In the next step, the DNA polymerase enzyme synthesizes a second DNA strand on each of the two original strands, using the free nucleotides in the solution. The annealed primer serves as a starting point (Fig. 18.1). New DNA is synthesized from the 3' end of each primer and extends in only one direction. The result is two helices where before there was only one. (DNA polymerase enzymes must have a 3' end from which to start synthesizing, and they can add nucleotides in only one direction; see DNA Replication [chapter 6].)
The process of denaturation, hybridization, and synthesis is then repeated, giving four helices (Fig. 18.1). A further round yields 8, then 16, then 32, and so on. Typical PCR procedures call for 25 to 40 rounds of amplification and yield huge numbers of molecules. Nearly all of the new DNA molecules have the primers as their ends and are the same length.
continued...In early PCR methods, new DNA polymerase had to be added after each denaturation step because the high heat necessary for denaturation destroyed the enzyme. Now, however, scientists have purified a DNA polymerase enzyme from a bacterium that inhabits hot springs (Thermus aquaticus). This enzyme, the Taq polymerase, remains active after being heated to 95°C and does not need to be added after each denaturation step. The Taq polymerase has made it simple to automate PCR: all that is needed is a heater with programmable temperature cycles. "PCR machines," called thermal cyclers, have become a fairly standard piece of laboratory equipment (Fig. 18.2).
PCR can produce enough product DNA, even from a minute amount of template, to be visible in a gel after electrophoresis. Biotechnologists often use this technique to produce many copies of an interesting DNA segment for cloning. PCR is also used in DNA typing when only minimal samples are available (see chapter 24). And, since the PCR process depends on the specific base pairing of primers to the template DNA, PCR can be used as a diagnostic tool.
In PCR-based diagnosis, the primers are chosen so that they hybridize only to a specific portion of the target organism's DNA or so that the distance between the primer hybridization sites is unique to the target. To determine whether the target organism is present, PCR is performed on the sample. If no DNA in the sample can hybridize to the primers, no unique PCR product will be synthesized. On the other hand, if the desired target DNA is present, there will be a unique product representing the segment between the primer hybridization sites. Because PCR produces so many copies of the segment, the product DNA can easily be detected by a variety of methods, including agarose gel electrophoresis and simple staining.
The great advantage of PCR as a diagnostic tool is its ability to amplify rare DNA. The amplification allows tiny amounts of specific DNA to be detected. In many clinical specimens, the disease-causing organism is present in very low numbers. PCR can reveal the presence of these rare organisms without taking time for culturing. PCR also permits the use of very small specimens. This advantage has made PCR an invaluable tool in studying ancient DNA samples. Researchers can use minute samples of museum specimens to produce enough DNA for analysis, thus preserving the original specimen essentially intact. Because the products of PCR are not usually designed to be longer than 2,000 base pairs or so, PCR also works well on samples in which the DNA has undergone extensive fragmentation. (For more information on the use of PCR to study ancient samples, see the reference below.)
Below is a paper PCR activity you can do with your students. Although real PCR primers are at least 16 nucleotides long (to provide greater specificity), the paper primers are only 5 nucleotides long. In addition, the segments amplified by PCR are usually hundreds of nucleotides long, whereas this activity amplifies a short segment. However, this paper model very accurately demonstrates the steps of PCR and shows how a specific DNA segment can be amplified from a single copy. The second part of the activity illustrates how PCR is used as a diagnostic tool.
Page 1