19.1.8 Polymerase Chain Reaction

• Polymerase chain reaction (PCR) is a common molecular biology technique used in most applications of gene technology, for example, DNA profiling (eg. identification of criminals and determining paternity) or genetic engineering
• It can be described as the in vitro method of DNA amplification
• It is used to produce large quantities of specific fragments of DNA or RNA from very small quantities (even just one molecule of DNA or RNA). By using PCR scientists can have billions of identical copies of the DNA or RNA sample within a few hours
• The PCR process involves three key stages per cycle. In each cycle the DNA is doubled so in a standard run of 20 cycles a million DNA molecules are produced. The three stages are undertaken in a PCR instrument (or thermal cycler) which automatically provides the optimal temperature for each stage and controls the length of time spent at each stage

• Each PCR reaction requires:
  • Target DNA or RNA being amplified
  • Primers (forward and reverse) – these are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied. They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands
  • DNA polymerase – is the enzyme used to build the new DNA or RNA strand. The most commonly used polymerase is Taq polymerase as it comes from a thermophilic bacterium Thermus aquaticus which means it does not denature at the high temperature involved during the first stage of the PCR reaction and secondly, its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
  • Free nucleotides – used in the construction of the DNA or RNA strands
  • Buffer solution – to provide the optimum pH for the reactions to occur in

• The three stages are:
  • Denaturation – the double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together
  • Annealing – the temperature is decreased to between 50 - 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
  • Elongation / Extension – the temperature is increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules

The substances required for polymerase chain reaction to occur and the three key stages of the reaction