Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is a technique widely used in molecular biology for analyzing and testing DNA. The DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication contributed to the name for this technology. The PCR process sets in motion a chain reaction in which the DNA template is exponentially amplified (copied). PCR allows scientists and researchers to amplify a limited amount of DNA so that millions or more copies of the same DNA piece can be generated for study or testing. The technology makes analysis and testing of DNA possible. Therefore, the PCR technology is widely used in both the DNA research and testing fields.
The PCR is commonly carried out in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction. The PCR occurs in the following steps:
Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that need heat activation by hot-start PCR.
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: At a temperature around 72°C, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
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