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Thermal Cyclers

Thermal Cyclers


Thermal Cyclers

A scientist at work in a modern molecular biology laboratory might be amazed to discover that in the early days of the polymerase chain reaction (PCR), the cycles were controlled manually. Each stage of the cycle was carried out on the bench top by laboriously raising the temperature and meticulously monitoring the mixture so as to not over shoot the required optimal range. The equipment consisted chiefly of a few water baths or heating blocks set to the required temperatures, and the vials with the PCR mix would be passed between them. Any mistakes made in setting the correct temperature could mean that the whole experiment would have to be restarted because of incomplete annealing or non-specific binding, or a host of other potential problems. Unfortunately, this would not be discovered until the end of the thirty-plus thermal cycles.

Thankfully, the thermal cycler was developed soon after the refinement of the PCR technique. The first machines are described in the literature in 1986, and they are a far cry from the water baths employed by the enterprising scientists of the 1970s and 80s. Essentially, a thermal cycler is capable of heating and cooling the DNA-containing mixture to the required temperatures with a high degree of accuracy. Solutions need to be not only rapidly cycled through the temperature ranges, but also accurately held at the correct conditions very precisely.

Steps of PCR

The general steps of PCR are roughly the same, although precise details may vary depending on the exact variation of the technique which is being used.

Firstly, there may be an initial heating step which is used to prevent mis-priming and to activate the Taq polymerase enzyme in the solution. This requires heating up to 95 degrees C for 5 minutes.

Denaturation: This stage of the cycle involves denaturation of the DNA molecules in the mixture. This requires heating up to 95 degrees C. The tertiary and secondary structure of the helix are lost and, at these high temperatures, the double bonds holding the base pairs together break. As long as this temperature is maintained, the DNA within it remains denatured. It can then serve to act as a template for new strands to form.

Annealing: As the temperature is lowered to around 60 degrees C, the primers present in the mixture are able to attach to the template strands. The exact temperature of the annealing stage is very important and can determine the success or failure of the whole reaction. Correct annealing of the primers is essential for duplication to occur. The temperature can be determined using simple calculations and there are computer programmes which enable this calculation based on the number of bases in the designed primers. It is usually roughly between 1 and 5 degrees C lower than the lower melting temperature of the two primers.

Extension: The temperature is now raised to 72 degrees C. This is the effective working temperature of Taq polymerase. This works to attach complementary nucleotides to those present in the denatured strands of DNA using the primers as the starting points. The results of this extension process are that two completely identical strands of DNA are produced.

Each time the cycle is repeated, the quantity of DNA strands is roughly doubled. And, as the fabled inventor of the game of chess, who asked for the grains of rice to be doubled for each square of the chess board, knew, this exponential growth soon results in billions of copies.

Variations of Thermal Cyclers

At its heart, a thermal cycler is a metal block which houses the PCR tubes. This is rapidly heated by means of a heating element, or bulb and cooled via a fan. Most thermal cyclers can be programmed with specific temperatures and cycle timings. Many can hold hundreds of programmes for different primers and experimental conditions.

When choosing a thermal cycler it is possible to become overwhelmed by the range and variety of different machines available to purchase. It is best not only to consider the experiment in hand, but to choose one which will provide enough flexibility to be able to deal with new developments to the PCR techniques and different variations which may be needed for the experiment.

It is best to look at the space which is available in the laboratory. A thermal cycler with a large footprint might take up unnecessary and valuable space. The position of the machine is also very important. It must be close enough to the PCR workspace to permit easy working without the problems of cross contamination.

For a small lab with a few workers, a simple personal machine might be the best choice. It is more economical to run a few tubes in a smaller machine. Larger labs might require high throughput thermal cyclers with more than one block. It is possible to get blocks which hold 96 well plates as well as 384 well. In more advanced equipment, each block can be controlled separately so that different experiments can be run at the same time.

Gradient thermal cyclers provide different temperatures across the same block. So, for example, if the correct annealing temperature is not known, a gradient can be used around a range to see which temperature yields the best results. This is a much more efficient way to find the temperature than having to run several experiments and allows for fast and effective PCR optimisation.

Many advanced thermal cyclers have their own built in computers, or a laptop or desktop computer is provided with the purchase which has dedicated software to make it easier to programme the cycles. They can hold many different settings which can be permanently programmed into the memory so that the same information does not need to be entered each time.

It is a wise decision to choose a machine which will be easy to use. It should be quite intuitive and new staff should not need to be extensively trained to use it. An eye should also be kept on the use of consumables. It is best to opt for a machine which can adapt to the consumables already in use within a lab rather than having to purchase a whole new range.