PCR and Thermalcycling

1. Understand the process of Polymerase Chain Reaction and how the thermalcycler helps carry out that process
2. Know the basic thermalcycler files

    Polymerase Chain Reaction (PCR) has had a temendous impact on the field of Molecular Biology, as significant as the initial use of restriction enzymes. The Taq Polymerase (and DNA Polymerase from other thermophilic bacteria) allows rapid amplification of a specific DNA sequence. However, the thermalcycler is as important to this process as the Taq Polymerase.  This machine produces rapid, sharp temperature changes for each othe three steps in the PCR cycle. The thermalcycler can be programmed to suit the needs of the experimenter.
 

The Step-Cycle File:
 
    The step-cycle file is used for DNA amplification. The typical step-cycle file has three segments. Usually, the first segment is used for template denaturation or melting (separation of double-stranded DNA). The second segment is used for primer annealing, and the third step is used for primer extension (DNA synthesis). Each cycle in the step-cycle file can be repeated up to 99 times. Usually, PCR is run for 20-30 cycles.

Thermalcycler Figure 1 shows two cycles of a step-cycle file that has three segments

    Each segment in a step-cycle file has a target temperature and a hold time, which is the length of time that the block will be held at the target temperature. The ramp time is the length of time that it takes the machine to move from one hold temperature to another. You cannot program ramp times; the machine heats and cools as quickly as possible. Thermalcycler manufacturers vie for the quickest ramp times in their machines.

For the three steps in the cycle, the usual target temperatures are:

• Denaturation = 94-96' C  (strand separation).
• Annealing = 45-60' C (the most variable; depends on the conditions of your experiment. As a general rule, the annealing temp should be 5' C below the Tm - melting temperature - of the DNA duplex).
• Synthesis = 72' C (the optimal synthesis temp for Taq).


    The Perkin-Elmer Thermalcycler used by the Molecular Biology lab has two additional features when programming a step-cycle file. Other PCR machines may not have these exact features.

1. after the three normal segments (denature, anneal, extend) then a "zero segment" is programmed with zero temperature and zero time. That tells the machine that the three segments programmed before the zero segment constitute one cycle.
2. "auto segment extension" will progressively lengthen the synthesis step by the programmed amount of time, usually 5-10 seconds. This means that each synthesis step gets progressively longer by 10 seconds for each cycle. This is one method to insure that partially-completed synthesis products have sufficient time to finish. See also the Time-Delay file below for another method to complete partial products.

 
The Time-Delay File:

    A time-delay file will take the heat block to a specific temperature and hold it at that temperature. The heat block is programmed for a certain delay time, which includes both the transition time and the incubation time. The delay time must be long enough to  include both this transition time as well as the desired incubation time.

The time-delay file is shown in Thermalcycler Figure 2

There are two common uses for the time-delay file:

• Before thermal-cycling, time-delay files are often used as high-temperature incubation files to destroy unwanted nucleases and proteases present in the DNA sample. This is particularly helpful if your DNA sample is not especially pure.
• After thermal-cycling, the time-delay file can be used as an additional extension or synthesis time for the Taq Polymerase. As the step-cycle file nears completion, there are often partially-completed synthesis products present. An additional time at 72' C enables the Taq to complete synthesis on these partial products.


The Soak File:

    A soak file is an incubation file in which the target temperature is reached as soon as possible and held indefinitely, until you stop the run by pressing the STOP button. Soak files are often used at the end of the run. The block is held at a cool temperature (5-15' C) to protect the samples from degredation, especially when the run goes overnight. It is the equivalent of putting the PCR machine in the refrigerator and coming in the next morning to get them.

A soak file is shown in the Thermalcycler Figure 3

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