PCR
(Polymerase Chain Reaction)

Medical Author: Frederick Hecht, MD, FAAP, FACMG
Medical Editor: Leslie J. Schoenfield, M.D., Ph.D.

• What is PCR (Polymerase Chain Reaction)?
• How is PCR done?
• What is the purpose of doing a PCR?
• How was PCR discovered?
• What is HCV PCR?
• What is RT PCR?

What is PCR (Polymerase Chain Reaction)?

PCR (Polymerase Chain Reaction) is a key technique in molecular genetics that permits the analysis of any short sequence of DNA (or RNA) without having to clone it. PCR is used to reproduce (amplify) selected sections of DNA. Previously, amplifying was done in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA. What is more, PCR uses the same molecules that nature uses for copying DNA:

• Two "primers" that flag the beginning and end of the DNA stretch to be copied;
• An enzyme called polymerase that walks along the segment of DNA, reading its code and assembling a copy; and
• A pile of DNA building blocks that the polymerase needs to make that copy.

How is PCR done?

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, 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:

1. Denaturation: At 94°C, the double-stranded DNA melts and opens into single-stranded DNA.
2. Annealing: At 54°C, hydrogen bonds form and break between the single-stranded "primer" and the single-stranded "template." (The template provides the pattern to be copied.) The more stable bonds last longer and on that little length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
3. Extension: At 72°C, the polymerase works best. As a result, the attraction, created by the hydrogen bonds, of the primers to the template is stronger than the forces breaking these attractions. The upshot is that bases complementary to the template are coupled to the primer.

Next: What is the purpose of doing a PCR? »

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Last Editorial Review: 6/4/2003