What are the fundamental steps of PCR?
Each PCR cycle includes a set of time- and temperature-controlled incubations. The incubations function to:
1. Denature: Denature the double-stranded target nucleic acid or DNA in a temperature range between 90 to 94 °C and separate them into single-stranded DNA.
2. Anneal: Anneal single-stranded amplimers, short oligonucleotides, at a temperature dependent on their calculated annealing temperature, usually in the range of 30 to 60 °C.
3. Extend: Extend primers using a thermostable DNA polymerase to synthesize new DNA strands by the addition of nucleotides to the 3’ end of each primer at a temperature around 70 to 74 °C. The DNA polymerase catalyzes the production of new complementary strands.