In the 1980s a method developed by American biochemist Kary Mullis revolutionized the molecular biology field. This method was PCR, for which he won the Nobel Prize for Chemistry in 1993. Polymerase Chain Reaction (PCR) is a method of making multiple copies of a strand of DNA. Based on the design on DNA replication inside a cell, he managed to create a method to bring about the in vitro version of it.
DNA replication is the process by which the cell produces a copy of the DNA, before it undergoes division. It creates an identical copy of the DNA strand, by splitting the double helix, and creating complimentary strands to each of the original strand. Therefore, each new double helix that is created has one old single strand and one newly synthesized single strand.
Before understanding how PCR works, it is better to understand the process of DNA replication. The process can be simplified as follows. First, the double helix structure is split up using enzymes to form 2 single stranded DNA. A small primer attaches to the end of each strand. These are essential in the process of replication, as the enzyme DNA Polymerase cannot create a new strand, but can rather, add nucleotides to an existing strand. There are 4 nucleotides – adenine, guanine, thymine, and cytosine. The polymerase adds nucleotides which are complementary to the template strand.
Once replication is complete, the primers are removed and breaks or gaps are filled in by the ligase enzyme. This concludes the process of DNA replication, resulting in 2 identical strands of DNA.
PCR follows a similar protocol, except it’s done outside the body, in a test tube, with certain parts modified to suit the conditions. The whole process occurs in 3 steps.
Step 1: Denaturation
As in replication, first the double helix needs to be broken. Unlike DNA replication, in PCR this separation is brought about by increasing the temperature rather than enzymes. The double helix is kept together by hydrogen bonds between the nucleotides of the 2 strands. This bond is weaker than the covalent bonding between the nucleotides of the same strand, and therefore is easier to break. A temperature of 95C achieves this.
Step 2: Annealing
As we know, for DNA polymerase to build a new strand, it needs a primer to attach onto the template strand. This property enables us to specifically choose which part of the DNA needs to be copied. Primers are synthetically made in the lab to attach to the ends of the desired portion. These primers can attach to the template strand at higher temperatures as compared to the two strands of DNA to each other. Therefore, the temperature is cooled to around 50-60C which is enough for the primers attach to the templates, but not enough for the double helix to form again.
Step 3: Extension
This is the final step of PCR. After attachment of the primers, the polymerase comes and forms the new strand by adding onto the primer. The DNA polymerase used by our body cannot be used in PCR. This is because the enzymes in our body have optimum functioning at our body temperature, which is 37C. However, at this temperature the 2 strands will form a double helix again, faster than the polymerase can act. In our body, this is prevented by the use of enzymes. In PCR therefore, the polymerase used is obtained from a bacterium called Thermus aquaticus. They are found in hot springs, and their polymerase is stable at higher temperatures. In fact, it works at an optimum temperature of 72C which is enough to keep the two strands from joining.
Uses of PCR
PCR is a very important part of molecular biology. It can be used to create thousands and millions of copies of a tiny amount of DNA, which is required in a number of situations
PCR is used during criminal forensics to salvage and save tiny bits of DNA found at crime scenes like those in hair, skin, nails, etc. Through PCR multiple copies can be made, making it easier to match samples, etc.
PCR is also important during the diagnosing of certain diseases. Sometimes, the number of organisms found in a patient is very small. In such cases, the DNA of the organism can by multiplied through PCR. This DNA can then be used to identify the organism. This is specially used when the organism cannot be easily grown in lab conditions.
PCR can also be used during genetic studies. For instance, if certain genes need to be inserted into another organism, the amount of the desired genes can be sufficiently created using PCR. This saves a lot of time and effort as compared to attempting to grow the organisms carrying the desired genes in the lab.
PCR is an important technique used by scientists used all over. It is essential for every aspiring geneticist, molecular biologist, biotechnologist, etc to be aware of this method and to understand the way it works.
- National Center for Biotechnology Information
- The University of Utah
- The University of Queensland, Australia
- Amrita Vishwa Vidyapeetham