2.Annealing 2.Annealing - starter chain of nucleotides act as primers for new nucleotides to attach to. 5’ 3’ 5’ 3’ 5’ 3’
5’ 3’ 5’ 3. Extending 3. Extending – heat stable enzyme (usually Taq polymerase) joins free nucleotides to the primer complementary DNA strand (semi-conservative).
taq polymerase isolated from thermophilic bacteria live in high temperatures so enzymes adapted for heat not denatured in PCR machine
PCR Equipment Simple-to-use PCR machines called thermal cyclers
Polymerase Chain Reaction Cycle 1 Original DNASample Cycle 2 Cycle 3 Many cycles Exponential growth
The Process of PCR 1 Primer annealed DNA sample = target DNA 98 C for 5 min denaturation mRNA primers are annealed
The Process of PCR 2 Nucleotides Semi-conservative copies Thermally stable DNA polymerase binds new nucleotides at 60 ° C
Limitations of PCR Normally used for DNA sequences 5kb (kilobases) long – most genes are far longer than this Can only generate moderate amounts of DNA (a billion DNA molecules is not a lot!) taq Polymerase cannot proofread to eliminate mutations if the gene is unknown, we cannot make the appropriate primer – so PCR useless