Polymerase Chain Reaction:

An Introduction to PCR for Students in Molecular Biology Lab Courses

By: Kacie DeOms

10/21/13

NATURE AND SCOPE

The purpose of this description is to introduce the process of Polymerase Chain Reaction (PCR) to students who will perform PCR in a college-level, molecular biology laboratory course. Students will use this description to become familiar with PCR and understand the concepts behind the experiment they will be performing in lab. It is likely that the students will be tested on this information. The audience will already possess a basic understanding of DNA replication and the directionality of DNA. All information regarding DNA replication, such as the purpose of DNA polymerase, primers, or dNTPs, should be a review for the audience. This description will provide enough detail for students to understand the essential steps and materials of PCR. Only basic PCR will be included in this description. Optimization tactics and other versions of PCR (i.e. hot-start PCR, quantitative PCR, etc.) will not be included.

INTRODUCTION
Polymerase Chain Reaction (PCR) is a common molecular biology laboratory technique used to exponentially amplify a specific DNA sequence. Through the use of PCR, millions of DNA copies can be generated from a single copy of a target DNA sequence. Kary Mullis invented PCR in 1983. Its development was so important to molecular biology that Mullis earned the 1993 Nobel Prize in Chemistry for his work. Researchers need large DNA samples to perform experiments and tests. Before the creation of PCR, these large samples had to be naturally available. PCRs ability to amplify a single strand of DNA into a sample of adequate size revolutionized biological research. PCR is used in medical, biological, and forensic settings for a variety of applications. In forensic science, PCR is used to create genetic fingerprints. These fingerprints are used in paternity testing and the identification of criminals. In medical fields, PCR is used to detect and diagnose hereditary and infectious diseases. In research labs, PCR is used for the analysis of genes, DNA sequencing, and other purposes. PCR is performed in three major steps: denaturation, annealing, and elongation (also called extension). These steps are repeated in this order for 20-40 cycles depending on the reaction performed and reagents used. A thermocycler (also called a PCR machine) executes each of these steps at a different temperature. In the denaturation step, the template DNA strands are separated. In the annealing step, primers attach to the DNA. In the elongation step, the DNA is replicated, creating a new double-stranded DNA sample. After each cycle, the newly

synthesized DNA is used as template DNA in the following cycle. This allows for an exponential increase in DNA through the cycles of PCR. Figure 1 provides an overview of PCR.

Figure 1: Overview of PCR (Source: New England BioLabs. pcr.jpg)

MATERIALS
PCR reactions are carried out in PCR tubes in the thermocycler. These tubes are small (about 0.2-0.5mL) and thin-walled to allow for the rapid heating and cooling of the PCR reaction. The following components should be included in a PCR reaction: DNA template. Template DNA contains the target region to be amplified. Two Primers. There should be one primer complementary to the 3 end of the sense strand and one primer complementary to the 3 end of the antisense strand of the target DNA region. DNA polymerase. The polymerase chosen should have an optimum temperature of approximately 70C. (Taq polymerase is used most often.)

dNTPs (deoxynucleotide triphosphates). These dNTPs are the building blocks of DNA that the DNA polymerase will use to synthesize new DNA strands. Buffer. A suitable buffer should be used to create an environment for optimum DNA polymerase activity.

Figure 2: PCR tubes and Taq DNA polymerase (Sources: POCD Scientific and Geneaid. PCR-photo.jpg and Taq.jpg)*

STEPS OF PCR
Denaturation In DNA replication, each strand of the double-stranded template is replicated. This results in two complete double-stranded DNA molecules. Each new molecule has one strand from the original helix and one new strand. To synthesize a new strand, the double stranded template must first be separated into two single-stranded molecules. In the denaturation step, the double-stranded template DNA is separated to allow DNA replication. For this step, the thermocycler heats the reaction to 94-98C for 20-40 seconds. This high temperature disrupts the hydrogen bonds that hold the DNA strands together, resulting in two single-stranded DNA molecules.

Figure 3: Denaturation Step (Source NCBI. PCR_principle1.gif)*

Annealing Primers are necessary for DNA polymerase to initiate DNA replication. In the annealing step, the primers are attached to the single stranded DNA. For the primers to anneal to the template DNA strands, the reaction temperature is lowered to 50-65C for 2030 seconds. The specific annealing temperature should be 3-5C lower than the optimal annealing temperature of the primers. Since the primers are complementary to the 3 ends of the single-stranded template DNA, they will form hydrogen bonds with the template at this lower temperature. Once the primers are annealed, DNA polymerase attaches to the primertemplate DNA hybrid and begins DNA replication.

Figure 4: Annealing Step (Source NCBI. PCR_principle1.gif)*

Elongation/Extension In the elongation step, DNA polymerase completes replication of the template strand. This results in two double-stranded DNA molecules. The temperature of the elongation step is chosen based on the optimum temperature of the DNA polymerase chosen for the reaction. The optimum temperature for Taq DNA polymerase (the most common polymerase used for PCR) is 75-80C. The time of the elongation step depends on the DNA polymerase used and the length of the target DNA region. To perform elongation, the DNA polymerase attaches the dNTPs to the 3 end of the primer, extending the new DNA strand in the 5 to 3 direction. DNA polymerase continues to add dNTPs until the new strand is completed (has reached the end of the target region). Assuming there is a sufficient amount of all reagents, the number of double-stranded target DNA molecules is doubled after each elongation step. This leads to an exponential increase in target DNA molecules after a number of cycles.

Figure 5: Elongation/Extension Step (Source: NCBI. PCR_principle1.gif)*

CONCLUSION
The steps of denaturation, annealing, and elongation mimic the process of DNA replication. By repeating this cycle 20-40 times, scientistsand studentscan create large samples of DNA targets from minimal amounts of template DNA. This feat was unheard of just 30 years ago! When Kary Mullis developed PCR by applying temperature changes to DNA replication, he changed molecular biology forever. Many advancements in research, medicine, and forensic science have been made possible by PCR, and many more are sure to come.

References PCR. (n.d.). In National center for biotechnology information. Retrieved October 14, 2013, from http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml Polymerase chain reaction. (n.d.). In DNA learning center. Retrieved October 14, 2013, from http://dnalc.org/resources/animations/pcr.html Polymerase chain reaction (n.d.). In Wikipedia. Retrieved October 14, 2013, from http://en.wikipedia.org/wiki/Polymerase_chain_reaction

Figure Notes *image modified by author