Lecture 3 - PCR

Question 1

What is PCR

Answer 1

an in vitro technique that allows the amplification of a DNA
fragment from DNA template

Question 2

What are the uses of PCR

Answer 2

Research: Detection of specific genes, sequences. Species id.
Medicine: Identification of pathogens (ex: virus!)
Biotechnology: Recombinant systems
Other: Forensics, paternity test, etc

Question 3

What is PCR based on?

Answer 3

the ability of the DNA polymerase to
synthesize a new strand of DNA complementary to the a
template strand

Question 4

What is DNA synthesis

Answer 4

sequential addition of deoxyribonucleotides to the 3ʹ-OH end of a polynucleotide chain, the primer strand, that is paired to a second
template strand

Question 5

PCR stages

Answer 5

Initiation
Denaturation
Annealing
Extension

Question 6

What is initiation (PCR)

Answer 6

The preparation of the reaction mixture, which includes the DNA template, primers, dNTPs (deoxynucleotide triphosphates), DNA polymerase (usually Taq polymerase), and a buffer solution.

This setup prepares the system for the thermal cycling process

Question 7

What is Denaturation?

Answer 7

The reaction mixture is heated to a high temperature (usually 94–98°C) to break the hydrogen bonds between the complementary DNA strands, resulting in two single-stranded DNA templates.

Purpose: Separates the DNA double helix into single strands to allow primer binding in the next step.

Question 8

What is primer annealing?

Answer 8

The reaction is cooled to a lower temperature (typically 50–65°C) to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates.

Purpose: The primers provide starting points for DNA synthesis. The specific temperature depends on the melting temperature (Tm) of the primers.

Question 9

What is Extension?

Answer 9

• The temperature is raised to the optimal working temperature of the DNA polymerase (usually 72°C for Taq polymerase). The enzyme extends the primers by adding complementary dNTPs to the template DNA strand, synthesizing new DNA.
• This step results in the elongation of the DNA strands, doubling the amount of target DNA with each cycle.

Question 10

What is amplification for in pcr

Answer 10

• the cycle is repeated 20–40 times, exponentially amplifying the target DNA region
• ensures that there is enough DNA to work with for accurate analysis, identification, and experimentation, especially when the starting material is scarce or degraded

Question 11

PCR in the lab material and equipment

Answer 11

• Pipettes / tips / ice / gloves
• Eppis and PCR tubes
• PCR instrument
• Electrophoresis

Question 12

PCR lab - Reaction mix

Answer 12

DNA template
DNA polymerase (thermally tolerant)
DNA polymerase reaction buffer
Mg2+ [MgCl2]
dNTPs [dATP, dCTP, dGTP, dTTP]
H2O
2 primers (forward and reverse)

Question 13

What DNA polymerases are used in PCR

Answer 13

• Thermus aquaticus -thermophilic bacterium
  (Taq polymerase)
• Pyrococcus furiosus - Pfu Polymerase -Proof reading activity (3‘→ 5‘
  exonuclease) – higher accuracy

Question 14

Why is MgCl2 used in PCR

Answer 14

• activates DNA polymerase, stabilizes dNTPs, and ensures proper primer-template interaction.
• Proper Mg²⁺ concentration is critical for efficient, specific, and high-fidelity amplification of the target DNA

Too little Mg²⁺: Low amplification efficiency or no product.
Too much Mg²⁺: Non-specific binding of primers and amplification of non-target sequences, leading to low specificity and primer-dimer formation.