Polymerase Chain Reaction, or PCR, is a laboratory method used to make millions or billions of copies of a specific DNA segment. It matters because many experiments and medical tests need more DNA than a sample naturally contains. PCR is used in disease testing, forensic identification, ancestry studies, cloning, and basic genetics research.
The method is powerful because it can target one chosen region of DNA instead of copying an entire genome.
PCR works by cycling a DNA sample through carefully controlled temperatures in a thermal cycler. High heat separates the two DNA strands, cooler temperatures allow primers to bind to the target sequence, and a heat stable DNA polymerase builds new DNA strands. Each cycle can approximately double the number of target DNA copies, so amplification is exponential.
The three main steps are denaturation, annealing, and extension, repeated about 25 to 40 times.
Key Facts
- Denaturation separates double stranded DNA into single strands at about 94 to 98°C.
- Annealing lets primers bind to complementary DNA sequences, often around 50 to 65°C.
- Extension occurs when Taq polymerase adds nucleotides to primers, usually at about 72°C.
- Ideal doubling follows N = N0 × 2^n, where n is the number of PCR cycles.
- A primer provides the free 3 prime OH group needed for DNA polymerase to begin synthesis.
- PCR requires template DNA, two primers, DNA polymerase, dNTPs, buffer, Mg2+, and a thermal cycler.
Vocabulary
- PCR
- PCR is a technique that amplifies a selected DNA sequence through repeated temperature cycles.
- Denaturation
- Denaturation is the PCR step in which heat separates double stranded DNA into single strands.
- Annealing
- Annealing is the PCR step in which primers bind to complementary sequences on the single stranded DNA templates.
- Taq polymerase
- Taq polymerase is a heat stable DNA polymerase that copies DNA during the extension step of PCR.
- Primer
- A primer is a short DNA sequence that marks the starting point for DNA synthesis.
Common Mistakes to Avoid
- Using only one primer is wrong because PCR needs a forward and reverse primer to define both ends of the target region.
- Setting the annealing temperature too low is wrong because primers may bind to similar but incorrect sequences, producing nonspecific DNA products.
- Forgetting that DNA polymerase extends only 5 prime to 3 prime is wrong because primer orientation determines whether the target region is copied correctly.
- Assuming amplification is always exactly doubled every cycle is wrong because real PCR efficiency can drop due to limited reagents, enzyme performance, or product reannealing.
Practice Questions
- 1 A PCR begins with 10 copies of a target DNA sequence. Assuming perfect doubling, how many copies are present after 20 cycles?
- 2 A 25 microliter PCR reaction needs primers at a final concentration of 0.5 micromolar. If the primer stock is 10 micromolar, what volume of primer stock is needed for one primer?
- 3 A PCR gives many unexpected bands on a gel. Explain how annealing temperature and primer design could cause this result.