PCR (Days 2–3)

What is PCR?

PCR (Polymerase Chain Reaction) is a laboratory technique used to amplify a specific DNA sequence, producing millions of copies from a small amount of template DNA. It relies on repeated cycles of denaturation, annealing and extension.

What is the purpose of PCR?

PCR is used to amplify a target DNA sequence for applications such as cloning, sequencing, diagnostics and gene analysis.

What are the five essential components of PCR?

PCR requires template DNA, forward and reverse primers, thermostable Taq DNA polymerase, dNTPs and a reaction buffer containing Mg²⁺ ions.

What is the function of the DNA template in PCR?

The DNA template contains the target sequence that will be copied during PCR. It provides the original strand from which new DNA molecules are synthesized.

What is the function of primers in PCR?

Primers are short single-stranded DNA sequences that bind to complementary regions on the template DNA. They provide the free 3′-OH group that allows Taq polymerase to begin DNA synthesis.

Why are two primers used in PCR?

A forward primer binds to one DNA strand and a reverse primer binds to the opposite strand. Together they define the beginning and end of the DNA fragment to be amplified.

What is the function of Taq DNA polymerase?

Taq polymerase synthesizes new DNA strands by adding complementary nucleotides to the 3′ end of each primer. It is heat stable and remains active after repeated heating.

Why is Taq polymerase preferred over normal DNA polymerase?

Taq polymerase is isolated from Thermus aquaticus and remains active at high temperatures used during PCR, unlike ordinary DNA polymerases that would denature.

What are dNTPs?

dNTPs (dATP, dTTP, dCTP and dGTP) are the building blocks used by Taq polymerase to synthesize new DNA strands.

Why is Mg²⁺ included in the PCR buffer?

Mg²⁺ acts as an essential cofactor for Taq polymerase. Without Mg²⁺ the enzyme cannot catalyse DNA synthesis efficiently.

What happens during the denaturation step?

The reaction mixture is heated to 94–98°C, breaking the hydrogen bonds between complementary DNA strands and producing single-stranded templates.

What happens during the annealing step?

The temperature is lowered to approximately 45–65°C, allowing primers to bind specifically to complementary sequences on the template DNA.

What happens during the extension step?

The temperature is raised to 72°C, the optimum temperature for Taq polymerase, which synthesizes new DNA strands by adding dNTPs to the primers.

Why is 72°C used during extension?

72°C is the optimum working temperature for Taq DNA polymerase, allowing rapid and efficient DNA synthesis.

How many DNA copies are produced after each PCR cycle?

Each PCR cycle approximately doubles the amount of target DNA, resulting in exponential amplification.

Why is PCR considered an exponential process?

Because each newly synthesized DNA molecule becomes a template in the next cycle, causing the amount of DNA to double with every cycle.

What is primer melting temperature (Tm)?

The melting temperature is the temperature at which approximately half of the primer-template complexes separate into single strands. It helps determine the annealing temperature.

How is primer melting temperature calculated?

Tm = 4(G + C) + 2(A + T).

Why should both primers have similar melting temperatures?

Primers with Tm values within about 5°C of each other anneal efficiently under the same PCR conditions, improving amplification.

What is a GC clamp?

A GC clamp is the presence of at least two G or C bases within the last five nucleotides at the 3′ end of a primer. It improves primer stability and binding.

Why are G and C bases more stable than A and T?

Guanine and cytosine form three hydrogen bonds, whereas adenine and thymine form only two, making GC-rich regions more stable.

What is the purpose of a positive PCR control?

A positive control contains a known DNA template and confirms that the PCR reagents, primers and cycling conditions are functioning correctly.

What is the purpose of a negative PCR control?

A negative control contains all PCR reagents except template DNA. It confirms that the reagents are free from DNA contamination.

What does a band in the negative control indicate?

A band in the negative control indicates contamination of the PCR reagents or laboratory environment with DNA.

What could happen if the annealing temperature is too low?

Primers may bind non-specifically to similar DNA sequences, producing unwanted PCR products.

What could happen if the annealing temperature is too high?

Primers may not bind efficiently to the template DNA, resulting in little or no PCR product.

What would happen if primers were omitted from the PCR reaction?

Taq polymerase would have no starting point for DNA synthesis, so amplification would not occur.

What would happen if Mg²⁺ were omitted from the PCR reaction?

Taq polymerase would be inactive and DNA amplification would fail.

Why is PCR repeated for many cycles?

Repeating the cycle 25–35 times produces millions to billions of copies of the target DNA, making it easy to detect and analyse.