PCR

PCR

1. making multiple copies of DNA fragments - identical pieces of D.N.A

2. The D.N.A fragment to be copied is added to a thermocycler, along with D.N.A Polymerase, free nucleotides and primers

   • Temperature begins @ 95degrees.
     • This breaks the hydrogen bonds, and separates D.N.A strands.
       • Temperature is then reduced to 40 degrees.
         • This causes the primers to attach to complementary base sequences at the ends of the single strands
           • The temperature is then raised to 72 degrees.
             • This allows DN.A Poiymerase to attach, and make new strands of DNA. This happens as the free nucleotides attach by complementary base pairing. The new strands use the original as a template
               • DNA POLYMERASE - thermostable enzyme, doesn't get denatures by extremes of temperature
                 • The cycle ends, and the process is repeated, doubling the amount of D.N.A each time
   • PRIMERS - short sequences of single stranded nuclotides. With a specific base sequence,. They are complementary to the ends of the fragments to be copied.
     • Primers allow D.N.A polymerase to bind

3. in vitro cloning - OUTSIDE LIVING CELLS