Application:
• Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions
- The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
- Each reaction doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
The reaction occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
- Denaturation – DNA sample is heated (~90ºC) to separate the two strands
- Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied)
- Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)
Taq polymerase is an enzyme isolated from the thermophilic bacterium Thermus aquaticus
- As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing
- Taq polymerase extends the nucleotide chain from the primers – therefore primers are used to select the sequence to be copied
Summary of a Single PCR Cycle
Overview of a PCR Sequence – First 4 Cycles
