By Robert Hazen, George Mason University
DNA fingerprinting is now used routinely to set free the innocent and to convict the guilty. Our ability to manipulate and analyze tiny amounts of DNA rests on a simple and elegant technique. It is called the polymerase chain reaction, or PCR for short. And, this reaction relies on DNA’s ability to duplicate itself.
How PCR Works
PCR targets a specific short segment of DNA, and then makes countless copies of that segment and that segment alone.
Here’s how it works. We begin by preparing a solution that contains the DNA sample of interest; perhaps it’s the chromosomes from a single cell. The target segment of DNA is often a very tiny proportion of that total, maybe only a few dozen base pairs long. To the solution that contains that DNA, we add large amounts of three key ingredients.
First of all, we add individual DNA nucleotides: that’s the individual rungs of a DNA ladder, the A, C, G, and T, so that we can make lots of copies. Second, we add an enzyme called polymerase, and that helps to assemble DNA strands from nucleotides. That’s what all enzymes do; they help us perform chemical operations and speed them up, and polymerase speeds up the formation of DNA.
Finally, we have numerous copies of what are called short primer segments of DNA. These segments target, recognize and bond to only specific desired sequences of perhaps 20 base pairs long on specific DNA areas, specific chromosomes. For example, out of the whole three billion base pairs in human DNA, we might be able to target one short segment and just duplicate that segment over and over again.
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Heating and Cooling
This solution is heated to about 200° Fahrenheit—that’s just below the boiling point of water—and at that temperature the DNA double helix automatically splits apart into single strands. It doesn’t happen in nature, because we don’t normally get to 200° Fahrenheit, but in a solution we can do this.
Where before we had double helices, now we have two single strands. This mixture, which is separated, has single strands of DNA. It’s then cooled to 140° Fahrenheit, and that’s cool enough for two of the primer strands to bind to the very specific target segments on the two exposed DNA chains in the target gene.
If one can imagine, we have two separate strands of DNA, and two very short segments now become a double helix, in an otherwise single-stranded molecule. Polymerase triggers the growth of new DNA strands from that point, and we start making new segments from those primer spots. The result is two new double helices of the short segment, where before there was one.
Repeating the Process
Then, we repeat the process. We heat it back up to 200°. The two short segments of DNA that we formed, the two new segments, split apart, and now we have four exposed primer pairs. We cool it down, and we get four new double helices. We heat it up, and we have eight single strands. We cool it down, and we get eight double helices, and so forth and so on.
Each time we cycle the temperature, we increase by a factor of two the number of strands. Within a few hours, we can have millions of copies of the targeted sequence, while the rest of the cell’s genome remains in its original single strands, the two single strands that we started with. Given this large amount of a single gene, or a single segment of DNA, it’s often possible to use routine procedures to determine the specific DNA sequence of that particular segment.
PCR in Forensic Applications
This polymerase chain reaction has found many applications in science and in technology. The most publicized uses are the forensic applications, where one uses it to identify small amounts of DNA associated with a crime scene. If we have traces of blood, a few cells of skin, perhaps some semen or other cellular material, we can amplify specific segments of that DNA, and look to see the characteristics of that particular individual. We can then take a similar blood sample from the suspect and we can do the same sort of test. We can match up these characters to sequences, to see how close they are. If they’re identical, then we have a match.
PCR provides molecular biologists with a powerful tool to identify and understand genetic characteristics. It becomes a research tool in the hand of the molecular biologist. It has found routine use as a rapid screening technique for numerous genetic diseases also, such as cystic fibrosis, muscular dystrophy; for viral contaminants, such as HIV in blood; for tissue compatibility before organ transplants. Any time we’re trying to compare and contrast two pieces of genetic material, PCR provides a wonderful way of doing this quickly, cheaply, and efficiently.
Common Questions about How Polymerase Chain Reaction Helps in DNA Fingerprinting
At 200° Fahrenheit—that’s just below the boiling point of water—the DNA double helix automatically splits apart into single strands.
PCR has found many, many applications in science and in technology. The most publicized uses are the forensic applications, where one uses it to identify small amounts of DNA associated with a crime scene. If we have traces of blood, a few cells of skin, perhaps some semen or other cellular material, we can amplify specific segments of that DNA, and look to see the characteristics of that particular individual.
PCR provides molecular biologists with a powerful tool to identify and understand genetic characteristics. It is also used routinely as a rapid screening technique for numerous genetic diseases, such as cystic fibrosis, muscular dystrophy; for viral contaminants, such as HIV in blood; and for tissue compatibility before organ transplants.
