Three Temperature Cycles
Polymerase Chain Reaction Process
A key insight to the success of PCR as an in vitro DNA replication process which generates millions of specific sequence copies was a three-temperature cycle which accomplishes three parts of DNA replication; Denaturation of the double stranded template, Annealing of the primers to the single strands and Extension of new strand synthesis by DNA pol III.
Figure 1. The three temperature steps in a PCR cycle. Denaturation is first and occurs around 95 Celsius, then annealing at 60 Celsius, then elongation at 65 Celsius. There will be 25-40 cycles of this.
Created by M. Hanneman, 2021.
In general, a single PCR run will undergo 25-40 cycles. The first step for a single cycle is the denaturation step, in which the double-stranded DNA template molecule (Fig. 2) is made single-stranded (Fig. 3). The temperature for this step is typically in the range of 95-100°C, near boiling. The high heat breaks the hydrogen bonds between the strands but does not break the sugar-phosphate bonds that hold the nucleotides of a single strand together (Fig. 3).
Figure 2. DNA template in the PCR reaction chamber (test tube) is a double stranded molecule. The targeted sequence of nucleotides for this illustration is shown. The template DNA could be fragments from the chromosome of various lengths. The 5’ and 3’ orientation of both strands is shown.
Created by M. Hanneman, 2021.
Figure 3. Denaturation. The hydrogen bonds holding the strands of the double stranded template together are broken by heating the test tube. Two different primers and the individual A, T, G and C nucleotides are shown.
Created by M. Hanneman, 2021.
Thousands of copies of the single stranded primers and the individual nucleotides were added to the test tube prior to beginning the cycles. Both the primers and nucleotides will become part of the new DNA strands. The second step in the PCR reaction is to cool the temperature in the test tube to 45-55°C. This is the primer annealing step in which the primers bind to complementary sequences in the single-stranded DNA template. The two primers are called the forward and the reverse primer and are designed because their sequences will target the desired segment of the DNA template for replication (Fig. 3).
Figure 4. Attachment of the primers to the template strand. The second step of PCR is to cool the test tube which allows the forward and reverse primer to bind to the template.
Created by M. Hanneman, 2021.
The geneticist planning the PCR analysis must “design” the forward and reverse primers and then buy them from a vendor who can synthesize single stranded DNA that has a specific sequence and length. The two most important criteria for primer design are the following:
- One primer must have a sequence that complements one of the template strands and the other primer must be complementary to the other strand. BOTH strands need to be primed for the replication process.
- The primers must bind so that their 3’ ends are ‘pointing’ in the direction of the other primer. This ensures that the sequence between the primers is replicated in the PCR cycles.
Extension: The final PCR step is when the DNA polymerase enzyme reads the template and connects new nucleotides to the primer’s 3’ end, extending a new complementary strand of DNA (Fig. 5). The test tube is heated to around 75°C, optimizing DNA pol III activity and the newly synthesizing DNA strand is extended as the template strand is read by DNA pol III. The extension step will run for a few minutes and this step completes one PCR cycle. Completion of the final step (extension) of the first cycle of PCR results in two double stranded DNA copies from the original template DNA, doubling of the amount of DNA present.
Figure 5. Extension step performed by DNA pol III. The time allowed for this step determines how much of the template DNA pol III can read and replicate.
Created by M. Hanneman, 2021.
For cycle 2, the denaturation, annealing, and extension steps are repeated (Fig. 6 a, b, c). This time there will be twice as many DNA template molecules compared to what there was at the beginning of cycle 1. Copies are made by reading the original template and the copies made in the previous cycle. Therefore, if everything is working correctly, the DNA replication in the test tube is a chain reaction where at the end of a cycle, there is double the amount of that particular DNA sequence as what was found at the beginning of the cycle.
Figure 6a. Cycle 1 is complete; two double stranded DNA molecules are made from the original double stranded template.
Created by M. Hanneman, 2021.
Figure 6b. Denaturation step generates four single strand templates. Two strands are the 3’ to 5’ strand and two are the complementary 5’ to 3’ strand.
Created by M. Hanneman, 2021.
Figure 6c. Annealing and Extension. All templates are primed and four 3’ ends provide a place of DNA pol III extension.
Created by M. Hanneman, 2021.
Because thousands of copies of the forward and reverse primer are added at the start of PCR, all the single strand templates, both the original, the copies in cycle 3 and beyond, and the copies of the copies made from previous cycles will be primed for the extension step of the cycles.
Figure 7. Thermal cycler. Thermal cyclers are a common machine found in genetic research labs worldwide.
Thermal Cycler
When PCR was first invented, scientists used water baths set at different temperatures and transferred the test tubes by hand at timed intervals to run the PCR reaction. Once the technique became a proven technology for DNA analysis, engineers went to work to create PCR machines. The instrument which heats and cools the DNA samples is called a thermal cycler (Fig. 7). Each small tube or sample well in a plate contains all the chemical components needed for a PCR reaction. Adding a specific sample to the reaction mix provides the template DNA. A thermal cycler can be programmed for specific temperatures and the amount of time spent at each temperature. The engineered design of thermal cyclers to maximize the accurate replication of the targeted DNA in a small sample volume with the minimum amount of time can be critical in many applications of PCR.
Taq DNA polymerase
When Dr. Kerry Mullis ran the first PCR experiments, he needed to add a new sample of DNA pol III after each denaturation step. This was because the high temperature needed to denature the double stranded DNA template also denatured the DNA pol III protein structure. The DNA pol III enzyme commonly available to molecular geneticists was from E. coli bacteria and this enzyme had no stability at near boiling temperatures. Fortunately, biologists had been investigating Thermus aquaticus (Taq) a thermophilic eubacterium found in hot springs (Chien et al. 1976). The Taq version of DNA pol III does not easily denature in the hot temperatures required in PCR; plus, it has a good efficiency, able to add 60 base pairs/sec at 70°C. Like all other DNA polymerases, Taq DNA pol III cannot begin DNA replication without the addition of a starting primer.
Thus, the discovery of Taq DNA pol III and the commercial availability of this enzyme made PCR a more reliable and doable technology which hastened its application to science investigation and diagnostic testing.