The Discovery of PCR

In 1983, Kary Mullis was driving along a Californian mountain road late one night. As a molecular biologist, Dr. Mullis was imagining a better way to study DNA. This late-night thinking led to a revolutionary way to make laboratory copies of DNA molecules (Saiki et al. 1985, Mullis 1990). In the decades since, the polymerase chain reaction or PCR, has become the standard method used for detecting specific DNA or RNA sequences. Selling the equipment and reagent kits for PCR is a multi-billion-dollar business because DNA and RNA detection is critical information in medical application such as virus detection, crime scene investigations for detecting specific DNA sequences left by suspects, or genetic research investigations.

PCR is an In Vitro process; a series of chemical reactions that happen outside of a living cell. This laboratory technique is modeled after an In vivo process, the living cell’s natural ability to replicate (make copies of) DNA during normal cell cycles. Every living cell makes a duplicate copy of each chromosome before the cell is ready to divide.

Prior to both mitosis and meiosis cell division in multicellular organisms, and cell division in prokaryotes, the DNA inside the cell must be replicated (see lesson Mitosis and Meiosis and the Cell Cycle). This replication process generates the genetic information needed for either two cells that are genetically identical with the original cell or four gamete cells with half of the original cell’s genetic information. Like all processes in living cells, a collection of specific proteins works together to perform enzymatic functions which control and complete chromosome replication. The function of three of these enzymes is described below.

Helicase: This is one of the enzymes that unwinds the double stranded DNA molecule. The unwound single strands no longer hydrogen bond to their complementary strand but the sugar-phosphate bonds remain intact.

DNA Polymerase III (DNA pol III): This is the main DNA polymerizing enzyme. The enzyme reads the single strand as a template and places in the complementary deoxyribonucleotide. DNA pol III reads the template in the 3’ to 5’ direction (verbally called “three prime to five prime direction”) and builds the new strand in the 5’ to 3’ direction, adding the next nucleotide to the 3’ end by catalyzing sugar-phosphate bonding. DNA pol III illustrates the specificity of enzymes several ways; it reads and proofreads the placement of new nucleotides to insure accurate replication. It can only read and build in one direction. DNA pol III can only add nucleotides to a free 3’ end. This last specification means that DNA pol III cannot start the replication process on the single stranded template. A third enzyme needs to be part of the in vivo replication team.

Primase: Biochemists named this enzyme to describe its role in starting or priming the replication process. Primase is a special RNA polymerase. The enzyme reads the single stranded DNA template 3’ to 5’ and adds ribonucleic acid (RNA) nucleotides in the 5’ to 3’ direction. Once a few hundred RNA nucleotides are added, primase falls off the template strand and leaves the 3’ end that DNA Pol III needs to continue the process.

There are other enzymes that play an important role in in vivo replication. However, PCR works as an in vitro DNA replication process by using just one of these enzymes. Mullis imagined a chemical reagent and a temperature change step in the method that could perform the work of the other two enzymes. It should be noted that because Dr. Kerry Mullis had learned about the details of in vivo DNA replication, he could create this science changing in vitro method.

Before reading the description of PCR components and processes, watch the Polymerase Chain Reaction video on MediaHub to help you visualize the importance of each step.