DNA amplification using PCR

* Isolate a DNA sample from tissue.

* The sample DNA strand is taken in a test-tube, along with taq polymerase, primers, an
abundance of free nucleotide bases
* and magnesium ions/buffer solution.
(The test-tube is placed in a thermocycler)
** Step 1: Denaturation. Temperature is raised to 94 C for 1 minute to break the
hydrogen bonds between the double DNA strand, forming single strands.
* Step 2: Primer Binding/Annealing. Temperature is reduced to 54 C, so that the primers
bond to the template strands.
* Step 3: Extension. Temperature is raised to 72 C, which is the optimum temperature
for taq polymerase. The bases align with their complementary pairs and are bonded by the
enzyme.
* Cycle is to be repeated to get a considerable amount of copied DNA, preferably 35 cycles
producing about 34 billion copies.

Temperature Alterations:
o In the context of denaturation of DNA / 90 to 95 C / step 1
- if temperature too low, DNA strands will not separate / eq;
o In the context of primer annealing / 40 to 70 C / step 2
- if temperature is too high, idea of less annealing e.g. less binding of primers;
o In the context of extension / 70 to 80 C / step 3
- if temperature is too low, synthesis of new DNA strands not completed / eq;
o if temperature is higher than 95 C, the enzyme will denature / eq;
o *if temperature is lower than 95 C, less breaking of the hydrogen bonds, so less
template strands formed.