The Polymerase Chain Reaction (PCR)
Now to our third key concept, the lab procedure called polymerase chain reaction. The vast majority of molecular markers rely on the use of PCR (Mullis et al. 1986).
This process mimics the natural way in which the cell replicates its DNA. It provides a quick, inexpensive way of making a large number (millions) of copies of a specific DNA segment.
A sample of DNA that you wish to be copied (the "template") is mixed with DNA polymerase and short targeted priming sequences ("primers") . Then, cycling through various temperatures leads to the production of a copy of the part of the template DNA sequence between the two primers. Reiterating the cycle many times allows the new copies to serve as templates in the next round, resulting in an exponential increase in copy number of the target sequence.
eLesson by Deana Namuth entitled Polymerase Chain Reaction (2004) provides a step by step approach to the PCR. She summarizes her lesson as follows: The polymerase chain reaction laboratory technique is used in a variety of applications to make copies of a specific DNA sequence. This lesson describes how a PCR reaction works, what it accomplishes and its basic requirements for success. Examples of interpreting results are given. PCR's strengths, weaknesses and applications to plant biotechnology are explained.
Animation by Leah Sandall depicts the process of PCR. Each component involved in the process is outlined and described in this animation.
Closer Look at Primers
An important component of PCR is the primer(s), which are short sequences of DNA (typically 10-30 base pairs long) that help initiate the synthesis process and also determine exactly which region(s) of DNA will be amplified. The design of primer sequences exploits the complementarity property of the DNA molecule. In the example below, the target sequence to be amplified (the "template") is shown in blue, and a possible primer sequence (18 bases in this case) is shown in red. Keep in mind that the primer sequences can be located anywhere along the template sequence, but must flank the key area of interest (the target). Notice the pairing of G-C and A-T.
The molecular biologist will determine the best primer sequences for the particular experiment or application. Designing good primers involves understanding some other concepts of DNA synthesis:
- the melting temperature of the double-stranded DNA (to know what annealing temperature to use for your PCR)
- the stability and relative GC content (GC bonds are more stable than AT bonds, which affects the melting temperature)
- avoiding complementarity within the primer sequence, as this inhibits proper annealing
Fortunately there are a number of software programs to help you design your primers, many of which are freely available on the internet (see for example http://bioinfo.ut.ee/primer3-0.4.0/primer3/ ). Once you have determined the best sequence to use for your primers, they can be purchased from any DNA synthesis facility.