An Overview of the Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a method that selectively replicates desired DNA segments in vitro. It requires that we design two primers, each 20-30 base pairs long, that will hybridize to the 3’ ends flanking the desired DNA segment. The forward primer and reverse primer are anti-parallel and complementary to the nonsense and sense strand, respectively. For instance, if the nonsense strand of the DNA has the sequence 5’ TTG CCA GAT 3’, the forward primer is 5’ ATC TGG CAA 3’. Additionally, restriction enzyme recognition sites can be added onto the 5’ end of these primers to facilitate ligation of the target DNA into a cloning vector.

First, these primers, along with Taq polymerase and other co-factors, are added to the target DNA that has been heated to 90°C for two minutes. The high temperature separates strands of the target DNA. Second, the sample is cooled to 42 to 65°C for 30 seconds, allowing the DNA will anneal with the primers. The temperature is raised to 72°C (the ideal temperature for polymerase) so Taq polymerase can extend the primers. 1 minute per 1kb of amplicon at 72°C.The thermal cycling is repeated for 25-30 times. Each cycle denatures the DNA, anneals the primers, and then extends the primers. The target DNA doubles in amount after every cycle. A temperature of 72°C is set for 5 minutes to ensure all primers have been fully extended.

Taq polymerase is a heat stable polymerase that remains active even after heating to 90°C. In addition to Taq polymerase, primers, and the target DNA, MgCl2, buffers, and 100-200 µM of each dNTP is added. Higher concentrations of dNTPs allow for faster cycles, but lower concentrations will increase fidelity. Taq polymerase has no 3’à5’ exonuclease activity, so having sufficient fidelity is important. Mg2+ is a required cofactor for polymerization, coordinating the dNTPs in the Taq’s active site.

To make sure the PCR went well and the primers hybridized correctly, we either sequence the amplicon directly or digest it with restriction enzymes and run it on a gel.

In nature, restriction enzymes protect bacteria from viral infection by cleaving foreign, DNA at specific recognition sequences. Bacteria have methyl transferases that methylate its genome, protecting it from cleavage. In lab, restriction enzymes are used to cut DNA sequence of interest. Treating DNA with a specific restriction enzymes and then running it on a gel allows us to see where recognition sites are located, and how frequent they appear in our sequence of interest.

EcoRV, for example, cuts blunt ends at 5’ GATATC 3’. If our PCR amplicon has 6 of these sequences, the amplicon will be cut 6 different times, resulting in 7 different sizes of DNA molecules. We can then separate the 7 different DNA fragments and determine their size with PCR. Different DNA molecules will contain a different number of recognition sites, so they will have unique fingerprints. The more kinds of restriction enzymes you use, the higher resolution the fingerprints will have. The fingerprints of your amplicon can then be compared to the fingerprint of the known sample. If they show the same bands, the PCR was successful.

Gel electrophoresis is a technique that separates DNA based on size and shape. DNA is loaded onto an agarose gel. A negatively charged cathode and a positively charged anode at the bottom of gel will apply an electric field to the gel. Because of phosphates in its backbone, DNA will migrate towards the anode at the foot of the gel. Larger or bulkier molecules of DNA will move slower through the gel matrix than smaller ones, which reach the foot of the gel more quickly. After a given time, the relative size of DNA molecules can be visualized by the distances they moved through the gel.

The gel is made with 50 ml .5 x TBE, .5 g agarose, with 2.5 ml ethidium bromide. Ethidium bromide is a fluorescent dye that intercalates between the bases of DNA. When the gel is put in a UV light box and exposed to UV light, the ethidium bromide bound to the DNA will fluoresce and report where our DNA bands are. Each band represents a population of DNA of a specific size. The size of each digested fragment is then determined by comparing how much the bands traveled compared to the DNA of known sizes in the control standards.

Rf = distance band traveled / total distance of dye front

The leftmost lane is the ladder control, and the four lanes on the right are comparing different samples of DNA treated with restriction enzymes. Each band represents a DNA fragment of a specific size.