15.14: PCR

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce millions or billions of DNA copies within just a few hours. In a PCR reaction, a heat-resistant DNA polymerase enzyme amplifies the original DNA through a series of temperature changes inside an automated machine called a thermocycler.

PCR is a Versatile Method that Revolutionized Molecular Biology

Kary Mullis developed PCR in 1983, for which he was awarded the 1993 Nobel Prize in Chemistry. Being a relatively fast, inexpensive, and precise way of copying a DNA sequence, PCR became an invaluable tool for numerous applications, including molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation and sequencing, and genetic disease diagnosis.

The PCR Reaction Cycle

PCR mimics the natural DNA replication process that occurs in cells. The reaction mixture includes a template DNA sequence to be copied, a pair of short DNA molecules called primers, free DNA building blocks called deoxynucleotide triphosphates (dNTPs), and a specialized DNA polymerase enzyme.

PCR involves a series of steps at high temperatures, requiring a DNA polymerase enzyme that is functional at such temperatures. The most commonly used DNA polymerase is Taq polymerase, named after Thermus aquaticus, the bacterium from which the polymerase was initially isolated. DNA polymerase is unable to synthesize a DNA molecule from scratch, or de novo. Instead, DNA polymerase adds to short DNA molecules, called primers, which bind to the DNA template through complementary base pairing. The primers provide a free 3’ hydroxyl group to which DNA polymerase can attach new dNTPs. There are four types of dNTPs in a PCR, one for each nucleotide in the DNA molecule: dATP, dCTP, dGTP, and dTTP.

Each PCR cycle consists of three steps: Denaturation, Annealing, and DNA Synthesis.

1. Denaturation. The PCR cycle is initiated by heating the reaction mixture to a high temperature, causing separation of the DNA double helix into two strands. This “melting” process usually occurs at a temperature of 90°C­­–100°C.
2. Annealing. The reaction mixture is quickly cooled (usually to 50°C–65°C), allowing the two primers to bind to their complementary sequences on the template DNA strands.
3. DNA Synthesis (Primer Extension). The reaction mixture is heated again, this time to a temperature (usually 60°C–75°C) that allows DNA polymerase to extend the primers by adding dNTPs that pair with the bases in the template strand.

A typical PCR involves 20-40 repeated cycles of these three steps, occurring in the thermocycler. Since the number of DNA molecules is doubled in each cycle, the DNA is amplified exponentially.

Limitations of PCR

If the scientist wants to amplify a specific stretch of the genome, the scientist must know at least part of the target DNA sequence to design appropriate primers. Another potential issue is the nonspecific annealing of primers to partially similar DNA sequences, leading to amplification of non-target DNA. This issue can be controlled by optimizing the reaction conditions. Being a highly sensitive detection method, PCR is also vulnerable to contamination, and even trace amounts of contaminating DNA can cause misleading results. The DNA polymerases used in PCR can be prone to errors. If a mutation happens within the first few cycles, most of the amplified DNA will carry the mutation.

The goal of PCR, Polymerase Chain Reaction is to amplify a genetic sequence. In this example, a gene of interest will be amplified from purified DNA. In order to perform the amplification, a polymerase is required to synthesize the new DNA.

A primer, a short piece of single stranded DNA that shares homology to the gene of interest is also needed. Finally, single nucleotides called Deoxynucleoside triphosphates or dNTPs are used to make the new strand. The first step in PCR is to heat the mixture which denatures the DNA.

Next, the reaction is cooled and the primers will anneal to their homologous region. Once bound the reaction is heated to an optimal temperature for the polymerase. The polymerase then recognizes the primer DNA complex and begins synthesis of the new strand using the dNTPs in the solution.

The reaction is then heated and proceeds as described across additional cycles. Following the third cycle, there are eight total copies of the gene. After the fourth cycle, 16 copies.

And fifth cycle, 32 copies. The number of cycles will continue to grow exponentially. After 30 cycles, there are just over one billion copies.