Core Concepts
In this article, we learn all about the Polymerase Chain Reaction, better known as PCR, including its history, detailed mechanisms, and practical use.
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Studying DNA
Ever since biologists began to realize the importance of DNA, techniques to isolate and replicate DNA sequences have been crucial in biological research. Specifically, such techniques needed to generate high concentrations of a certain stretch of DNA, which scientists can analyze through light absorption, gel electrophoresis, and other methods.
For decades, the primary method of amplifying stretches of DNA involved a process biochemists call molecular cloning. However, this method often required days, if not weeks, to yield a concentrated sample of DNA. Thus, the demand among scientists remained high for the development of a new technique. This is where PCR, or Polymerase Chain Reaction, comes into play.
What was once a slow and costly process quickly became efficient, simple, and reliable. With PCR, scientists developed many new applications involving DNA, such as DNA fingerprinting, paternity testing, and viral infection testing.
Due to its importance today, it remains valuable to ask: what exactly is PCR? To understand how PCR revolutionized science, we need to take a closer.
What is PCR?
Like molecular cloning, PCR requires two important components:
- DNA Primers
- Taq DNA Polymerase
Unlike molecular cloning, however, PCR doesn’t require a bacterial strain or any live organism. Instead, DNA is amplified by purely chemical means.
Importantly, you do need to know some information about the sequence you wish to replicate. You have to know a small stretch of bases immediately before and after your desired sequence. For PCR to work, you need to engineer short sequences of single-stranded DNA (ssDNA) that correspond to these stretches. Biochemists call these stretches “primers” and you need two of them: one that base pairs with the DNA before your desired sequence and one that base pairs with the complementary DNA after your desired sequence. Thus, your second primer bonds to the DNA after your desired sequence on the other strand.
Along with your DNA primers, you also need a special enzyme called Taq DNA Polymerase. All organisms have some form of DNA Polymerase, which assembles strands of DNA using primers. However, Taq DNA Polymerase has a unique origin: a species of microbe that lives in the volcanic areas of Yellowstone. Accordingly, this form of DNA Polymerase is uniquely adapted to function at high temperatures, which allows for more rapid polymerization. Further, unlike most DNA Polymerases, the Taq variety does not denature at high temperatures.
Once you have both DNA primers and Taq DNA Polymerase, along with your desired sequence in a solution of free nucleotide triphosphates, PCR may occur. Your reaction mixture must pass through three distinct phases of temperature, and this cycle repeats multiple times to fully amplify your sequence.
PCR Phase 1: Denaturation
In the first phase, the temperature of your reaction mixture increases high enough for your DNA with your desired sequence to “denature” or “melt”. This simply means that the two strands separate. Note that at this stage, your desired sequence is embedded in a much larger strand of DNA. This could be as large as an entire chromosome, but it’s much more common to be a bacterial plasmid.
PCR Phase 2: Annealation
In the second phase, the temperature drops in your reaction mixture for “annealation” to occur. This involves the binding of your DNA primers to their respective sequences. Since the primers are much shorter than the strands, the longer strands bind much faster to the primers than to themselves.
PCR Phase 3: Extension
In the third phase, the temperature slowly increases which allows for Taq DNA Polymerase to bind to the primers. Then, the enzyme recruits freely floating nucleotide triphosphates, base-pairing with the complementary DNA strand, extending the primer along the length of the strand. Since all DNA Polymerases can only perform DNA replication in the 5’ to 3’ direction, the replicated DNA always includes either the desired sequence itself or its complement. Per double-strand of parent DNA, this step forms two single strands of slightly-shorter DNA that begin at a primer.
PCR Phase 4: Repeat
These same three temperature phases repeat dozens to hundreds of times to amplify the desired sequence. Once the second cycle begins, the slightly-shorter DNA formed in the previous cycle will bind to the opposite primer that produced it. Once replicated by the Polymerase, the resultant strand includes your desired sequence (or its complement) and the short sequences corresponding to your primers.
In later cycles, all subsequent primer bindings to this short sequence yield further stretches of the same length, with either the same sequence or its complement. Of course, further replication also occurs on your original parent DNA, but throughout multiple cycles, the amount of DNA with your desired sequence dwarfs that of any other DNA. With this, you have successfully amplified your desired sequence and produced a concentrated solution.
Applications of PCR
The real utility of PCR in everyday life comes from its ability to identify the presence of certain DNA sequences. If you want to test whether a given sample has a certain sequence of DNA, you can design primers that correspond to sections of that desired sequence. Then, when you perform PCR on that sample, the only DNA replication that occurs must involve your desired sequence; this is because Taq DNA Polymerase can only synthesize DNA strands from primers bound to a template strand.
This may seem abstract, but imagine you know the DNA sequence of some viral pathogen; you can design primers to selectively base-pair with that DNA. Or imagine you have a blood sample from a crime scene with a possible suspect; you can design primers that selectively bind to unique sequences (called short tandem repeats) from your suspect’s DNA. Or you could perform a similar procedure to determine parentage in the context of a paternity test.
Gel Electrophoresis
But how do we know if replication has occurred in our DNA of interest? To analyze our finished PCR reaction, we load the samples into an agarose gel and perform gel electrophoresis. This procedure involves placing the gel in a buffer solution with two large electrodes at either end. At the top, near our wells with our sample, exists a negatively charged electrode (or anode), and opposite exists a positively charged electrode (or cathode).
Once you load your samples and turn on your electrodes, your DNA will begin to move toward the cathode. This is because DNA has a negative charge at most pH levels due to its phosphate backbone. Importantly, smaller DNA fragments move faster through the gel than large fragments. After just a few minutes (less than 20), any small fragments will have moved significantly through the well while the parent strands remain basically in the same place. When imaged under UV light, you can visually see the banding of any small fragments of desired DNA.
