BSCI 1510L Literature and Stats Guide: How PCR works

In the previous section, general aspects of DNA replication were discussed.  We will now examine the details of a specific molecular biological tool based on DNA replication: the polymerase chain reaction (PCR).   PCR has revolutionized molecular biology by allowing the production of large quantities of specific DNA sequences from as small an amount as a few molecules of template DNA. 

The basic principle of PCR is as follows.  In each cycle of PCR, double-stranded DNA is melted (separated into single strands) at approximately 95°C.  The DNA is then cooled to about 60˚C so that specific primers anneal to complementary regions on the single strands of DNA.  At this temperature the kinetic energy of the DNA is low enough for the hydrogen bonds to form between the template strand and the primer.  The primers bracket the region that is to be amplified by specifying the location of the start of replication and by initiating that replication.  The DNA is then held at the optimal temperature for the activity of the DNA polymerase that replicates each template strand starting at the primers.  The cycle then begins again with the melting phase.  With each cycle, the number of amplified strands doubles, resulting in an exponential increase in the amount of the desired DNA (thus the term "chain reaction" in the name of the process).  The huge increase in number of copies is called amplification.

Central to the operation of PCR is Taq polymerase, a DNA polymerase derived from Thermus aquaticus, a bacterium isolated from a hot spring in Yellowstone National Park.  Unlike typical DNA polymerases, Taq remains stable at temperatures near the boiling temperature of water and has optimal activity at about 65°C.  This allows the DNA to be melted in each cycle without destroying the activity of the polymerase.  In order for DNA synthesis to proceed, magnesium ions must be present in addition to the four types of dNTPs which serve as raw materials (dATP, dCTP, dGTP, and dTTP). 

The high degree of specificity of the PCR amplification results from the primers used to initiate DNA synthesis.  PCR primers are generally about 20 bp long.  They are designed to have a sequence that is complementary to the two template strands at the 3' ends of the region to be amplified (Fig. 2).  After the primers anneal to the template strands, DNA polymerase replicates the template strand starting at the 3' ends of the two primers.  Under appropriate annealing temperatures, the primers will bind very specifically to their target sequence (if it is present in the sample), allowing a researcher to amplify a tiny "needle" of sequence from the huge "haystack" of an entire genome. 

Fig. 2. Stages in the first cycle of PCR (actual primers would generally be longer)

In the first cycle of PCR (Fig. 2), the complementary strands produced have indeterminate lengths because there is nothing to stop synthesis along the original template strands (other than the limited time before the temperature is raised again to 95 ºC). However, in the second cycle products complementary to these indeterminate-length daughter strands have a short, fixed length determined by the position of the two primers (Fig. 3). 

Fig.3. Proliferation of types of strands during the first two cycles of PCR

In subsequent cycles, indeterminate-length strands continue to be produced at a constant rate from the original template DNA, but the short, fixed-length strands accumulate exponentially.  After a few cycles, the number of indeterminate-length strands becomes insignificant in comparison to the number of short strands flanked by the primer sequences.  Thus, at the end of the PCR run, one can assume that the DNA present is primarily composed of short, fixed-length segments bracketed by the primer sequences.

Although tedious, the temperature changes required for PCR can be produced by moving the samples repeatedly between fixed-temperature water baths.  However, most PCR is done in a thermocycler - an instrument that contains a heating/cooling block that can be programmed to produce a series of temperatures. It is interesting to note that PCR is a patented process and a part of the cost of the materials for this lab is a licensing fee to practice the process in an "authorized" thermocycler.