DNA can be manipulated using a number of molecular techniques in order to fulfil a wide array of biotechnological applications

• DNA can be amplified via the polymerase chain reaction
  

• DNA can be separated via agarose gel electrophoresis
  

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:

1. Denaturation – DNA sample is heated (~90ºC) to separate the two strands
   

2. Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied)
   

3. 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
  

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate and isolate DNA fragments based on mass / size

• DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
  

• Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
  

• Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
  

• Smaller samples are less impeded by the gel matrix and hence will move faster through the gel
  

• This causes samples of different sizes to separate as they travel at different speeds