The PCR usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps: denaturation, annealing and elongation (or extension) (Figure 5).
- The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturation. It breaks apart the hydrogen bonds that connect the two DNA strands;
- Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated;
- Time: usually 1-2 minutes, but up to 5 minutes. Also
certain polymerases are activated at this step (see hot-start
- After separating the DNA strands, the temperature is lowered so that the primers can attach themselves to the single-stranded DNA. This step is called annealing;
- The temperature depends on the primers and is usually 5°C below their melting temperature (Tm, 45-60°C, see 1.2.3 Primers). The wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random;
The annealing temperature can be approximated using the following formula:
4(G+C) + 2(A+T) - 5 = Annealing temperature (°C)
G+C and A+T are the numbers of the respective bases in the primer.
- Short-lived bonds are constantly formed and broken between the primer and the single-stranded template. The more stable bonds formed between exact matches of the primer and complementary DNA last a little longer;
- The polymerase attaches to this double-stranded DNA and starts copying the template;
- Time: 1-2 minutes.
- The DNA polymerase copies the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This is done at a higher temperature than the annealing step. This step is called elongation or extension;
- The primers having been extended for a few bases do not fall off at the higher temperature required for elongation as they have stronger hydrogen bonding to the template than the forces breaking these attractions;
- Primers that do not match exactly melt away from the template due to the higher temperature and are not extended;
- The nucleotides (complementary to the template) are added to the 3' end of the primers (the polymerase adds dNTP's from 5' to 3', reading the template in a 3' to 5' direction);
- The elongation temperature depends on the DNA polymerase. Taq polymerase elongates optimally at a temperature of 72°C;
- The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule, this step takes 1 minute per thousand base pairs;
- A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied. This step is typically 10-15 minutes long;
- Elongation of each strand continues past the primer
sites so that some of the flanking DNA is also copied. By cycle three there are
eight copies of the target DNA of which two are the desired product. After this
stage the number of the required PCR products increases exponentially and
becomes the major product of the reaction.
The DNA polymerase used in the first experiments was isolated from bacteria growing at temperatures of up to 73°C. These polymerases were destroyed by the high temperatures used in the denaturation step and so fresh enzyme had to be added at each cycle making PCR a labour intensive process. The discovery of bacteria living in hot springs led to the isolation of a polymerase that was thermostable. The first enzyme was isolated from Thermus aquaticus and is called Taq polymerase.
Another factor reducing the efficiency of the procedure was the fact that initially water baths set at the required temperatures were used and the reaction tubes had to be moved manually. Now many companies have designed and made PCR machines which can be programmed to carry out the steps as needed. Thus PCR has become automated and is widely used.
The animation shows PCR in action.
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The PCR product can be identified by its size using agarose gel electrophoresis. The gel is stained with ethidium bromide (or another dye) which binds to the DNA and then visualised under UV light. In this way the DNA can be seen and the size of the fragments determined by comparison with a DNA marker ladder (Figure 6).