polymerase-chain-reaction

Polymerase chain reaction (PCR)

• 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 an 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

Requirements of PCR reaction

• 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

• Primers define the region that is to be amplified by signalling 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

  • 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

Stages of PCR reaction

• 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