PCR Amplification of DNA
The polymerase chain reaction (PCR) is a DNA amplification technique that has revolutionized almost all aspects of biological research. The PCR technique was invented in 1984 by Dr. Kary Mullis while at Cetus Corporation. Mullis was awarded a Nobel Prize for his work in 1994. PCR allows for the amplification of a small quantity of DNA over one million-fold. The enormous utility of PCR is based on its procedural simplicity and specificity. Since the first application of PCR to diagnose sickle cell anemia, a large number of procedures have been developed. PCR has made amplification of DNA an alternate approach to cloning experiments. PCR is also used extensively in forensics, paternity/kinship testing, and the identification of human remains.
PCR amplification requires the use of a thermostable DNA polymerase. The most commonly used of these is Taq DNA polymerase, purified from a bacterium known as Thermus Aquaticus that inhabits hot springs. This enzyme remains stable at near-boiling temperatures. Also included in the PCR reaction are the four deoxynucleotides (dATP, dCTP, dGTP, and dTTP) and two synthetic oligonucleotides, typically 15-30 base pairs in length, known as "primers". These components, together with the DNA to be amplified, are incubated in an appropriate buffer that contains Mg2+. The primers are designed and synthesized to correspond to the start and end of the DNA to be amplified, known as the "template" or "target". If the template DNA is prepared from biological tissue, freshly isolated DNA will give the best amplification results. DNA extracted from older specimens may be degraded and therefore less suitable for amplification.
The PCR reaction mixture (which contains the Taq DNA polymerase, buffer, deoxynucleotides, primers, and template) is subjected to sequential heating/cooling cycles at three different temperatures. The three temperatures are the basis of the PCR process (Figure 1). In the first step, the template is heated to near boiling (92° - 96°C.) to denature or "melt" the DNA. This step, known as "denaturation" disrupts the hydrogen bonds between the two complementary DNA strands and causes their complete separation. In the second PCR step, the mixture is cooled to a temperature that is typically in the range of 45° - 65°. In this step, known as "annealing", the primers, present in great excess to the template, bind to the separated DNA strands. In the third PCR step, known as "extension", the temperature is raised to an intermediate value, usually 72°C. At this temperature the Taq DNA polymerase is maximally active and adds nucleotides to the primers to complete the synthesis of the new complementary strands.
The exact temperature and incubation time required for each step depends on several factors, including the length of the target DNA and GC content of the primer/template. In some cases, the annealing and extension steps may be combined resulting in a two step per PCR cycle.
The three PCR steps of denaturation, annealing, and extension constitute one "cycle" and result in a doubling of the template copies in the mix- ture. The process is typically repeated for 20-40 cycles. Theoretically, if the reaction is allowed to be repeated for (n) cycles, the number of copies of template DNA will be 2n following completion, as shown at the bottom of Figure 1. For example, one would anticipate one million-fold amplification after 20 cycles. In theory, this process could continue indefinitely. In practice, however, the amount of product reaches a maximum after about 40 cycles, due to the depletion of reaction components and loss of DNA polymerase activity.
One common problem that occurs during PCR is unwanted amplification products. These are due to contamination of the sample or nonspecific annealing of the primers. To reduce contamination, autoclaved tubes, pipet tips, and sterile water should be used. Gloves should always be worn when performing PCR.
To minimize unwanted PCR products due to nonspecific primer annealing, the primer concentration should be minimized, if possible. Another common technique is "hot start", in which the components of the PCR reaction are fully mixed only after the DNA is fully denatured above 94°C.
Following PCR, the amplified product is processed, depending on the objective of the experiment. In most cases, the DNA is subjected to either aga- rose or polyacrylamide gel electrophoresis. In DNA fingerprinting, used in criminal forensics, PCR-amplified DNA from the crime scene is compared to amplified DNA from suspects. In cloning experiments, the amplified DNA is typically further purified and ligated into the desired vector. In DNA sequencing experiments, the amplified DNA (usually radioactively labeled) is run on a thin polyacrylamide gel sequencing gel. The gel is then placed on a piece of film, which is activated by the radioactive molecules and developed to create an image of the DNA fragments in the gel. Alternatively, one may avoid the use of radioactivity by subjecting the sequencing gel to silver staining, in which silver ions bind directly to the DNA fragments.
The objective of this experiment is for students to gain hands-on experience of the principles and practice of Polymerase Chain Reaction (PCR).
This experiment has three modules:
Module 1: PCR Reaction Using a Thermal Cycler
Polymerase Chain Reaction Cycling
On the final cycle, the 72°C incubation can be extended to 5 minutes.
Module II: Separation of PCR Reactions by Agarose Gel Electrophoresis
Preparing the Agarose Gel
After the gel is cooled to 60 C
Loading DNA Samples
Standard DNA Fragments
Control reaction, 0 cycle
Reaction sample, 15 cycles
Reaction sample, 30 cycles
Running the Gel
Staining and Visualization of DNA
Module III: Size Determination of PCR Amplified DNA Fragment
The size of the PCR amplified DNA fragment can be extrapolated by its migration distance relative to the Standard DNA Fragments, for which the size of each fragment is known.
In each case, measure from the lower edge of the sample well to the lower end of each band. Record the distance traveled in centimeters (to the nearest millimeter).
 Lab adapted from Edvotek PCR Amplification of DNA Lab #330