3.2.2 Biotechnology

DNA tools

  • DNA can be amplified via the polymerase chain reaction (PCR)

  • DNA can be separated via agarose gel electrophoresis

 

PCR

  • PCR is an artificial method of replicating DNA under laboratory conditions

  • PCR has several stages and is repeated over and over to make copies. The repeated stages are carried out in a thermal cylinder

PCR : reaction mixture

  • DNA sample is placed into a vial with:

    • Primers (short pieces of DNA that are complementary to the base at the start of the DNA fragment to amplify it)

    • Taq DNA polymerase (enzyme to create new strand)

    • Free nucleotides (used to create new DNA strands)

Step 1

  • Denaturation - reaction mixture is heated for 1 minute at 90 o to break hydrogen bonds between nucleotides, separating the DNA strand

 

Step 2

  • Annealing - reaction is cooled to 50 to 60o for 20 seconds to allow primers to bind (anneal)  the DNA strands. Allow DNA polymerase enzyme to start to copy the strand

Step 3

  • Extension - increase temperature to 72 o for at last 1 minute. Optimum temp for Taq DNA polymerase enzymes. Taq DNA polymerase builds up complementary strands of DNA using free nucleotides

 

 

Gel electrophoresis

 

  • DNA is negatively charged

  • Longer fragments carry more weight so they don't travel as far as shorter fragments of DNA

  • Gel electrophoresis is used to separate and isolate DNA fragments bases 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 on each nucleotide

  • Smaller samples are less impeded by the gel matrix so they will move further and faster

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