My Fascination with Science - PCR Tests - 2. Some Basics

PCR Stands for Polymerase Chain Reaction. Think of it as a clever copy machine that can create amplified copies of a certain portion of a very dense page and it can produce millions and millions of them in a short time. Where PCR has revolutionized science is in making millions or billions of copies of portions of DNA (genetic material in organisms) quickly. Whenever you think about chemistry and "quickly" it will involve an enzyme, which accelerates chemical reactions. DNA Polymerases are enzymes that help synthesize DNA fast from their components.

Credit: Wikimedia

From Wikipedia - "DNA is is a molecule composed of two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses" . In very simple terms, DNAs are extremely long, consists of two strands and have millions of nucleotides.

Each of the two strands is wound as a helix and both are connected using what are referred to as "hydrogen bonds". As complex as they are, they are composed of just four bases (part of a nucleotide), called Adenine (A), Thymine (T), Guanine (G) and Cytosine (C). Further, the base in one strand of the helix dictates what is in the other - A & T are always paired and G and C are always paired. Each strand has a unique directionality to it that are opposite of each other. This is critical in the PCR process. I will talk about this in the next few posts.

Despite the fact that we have only four nucleotides and two unique pairs (four if you want to be strict), the number of combinations you can create when there are millions of them is huge and this is what gives rise to complexities and intersting variations in DNA. 

There are numerous applications where we need to concentrate on a small snippet of this humongously long chain. For example, this small snippet may be relevant to detecting particular diseases, or is unique to a criminal that needs to be matched against samples from a crime scene, or unique to a particular bacteria or virus which can be used to find if someone is infected with a specific disease. Scientists have identified such unique snippets in DNAs of various organisms by now thanks to rapid advances in technology. However, samples tend to have very small quantities of DNA and copying them to get millions of identical copies helps simplify further analysis. 

We have a DNA sample with millions of bases, but depending on the application, we are only intereted in a small snippet of it. Wouldn't it be cool if we can simply make copies of just that portion in enough quantities that it can be easily analyzed further? This is exactly what PCR does. It does this in three steps that are then repeated multiple times:

• As a zeroth step, understand clearly the nucleotide sequence of the snippet that you want to copy.
• Denaturation - where you basically separate the two strands of DNA by heating.
• Annealing - where you add synthesized fragments called Primers. One primer forms the second (complementary) helix at the beginning of the snippet of interest with one of the denatured strand and another which pairs up with the end of the snippet on the other. You make them attach this way by carefully cooling the system.
• Extension - This is where the polymerase swings into action by extending one end of the primer with nucelotides that has been added. At the end of one cycle, you will now have two double helical DNA, but with overhangs of single strand DNA, because of where the primers are and how the polymerase only extends in one direction.
• You repeat this process whereby during each cycle, you double the amount of DNA. But, the beauty of this process, as you will see later, is that after the second cycle, one of the DNA that will get copied this way will be just the snippet of interest. It turns out that after about 30 cycles you will likely have a billion copies, Yes A BILLION! And guess what, this whole process has been automated and each cycle only takes a few minutes each and the entire process can be completed in a few hours. 

Kary Mullis invented this and if you are interested, you can read about him here and I encourage you to watch this video. He invented this in 1983 and received Nobel Prize in Chemistry for this in 1993. I should caution you that there are many videos of him on YouTube, but be careful in choosing which one to watch. At least a couple I watched, while funny, got into how he doesn't believe that there is a global warming issue and I don't want tht to cloud his excellent contributions :)