As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step -- denatauration (alteration of structure), annealing (joining), and extension -- takes place at a different temperature:
- Denaturation: At 94 C (201.2 F), the double-stranded
DNA melts and opens into two pieces of single-stranded DNA.
- Annealing: At medium temperatures, around 54 C (129.2
F), the primers pair up (anneal) with the single-stranded "template" (The
template is the sequence of DNA to be copied.) On the small length of
double-stranded DNA (the joined primer and template), the polymerase attaches
and starts copying the template.
- Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.
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