Question

Which of the following statements regarding polymerase chain reaction is FALSE? a. Human DNA polymerase is used because it is the most accurate. b. A primer must be prepared with a complementary sequence to part of the DNA of interest. c. Repeated heating and cooling cycles allow the enzymes to act specifically and replaces helicase. d. Each cycle of the polymerase chain reaction doubles the amount of DNA of interest.

Short answer

a. Human DNA polymerase is used because it is the most accurate.

Step-by-step solution

Understand PCR

Polymerase Chain Reaction (PCR) is a technique used to amplify specific segments of DNA. It involves repeated heating and cooling cycles to denature the DNA, anneal primers, and extend the DNA.

Examine each statement

Carefully read each statement to determine if it accurately describes some aspect of PCR.

Evaluate Statement a

Statement a claims that human DNA polymerase is used because it is the most accurate. Human DNA polymerase is not used in PCR. Instead, Taq polymerase (from Thermus aquaticus) is used because it is heat-resistant.

Evaluate Statement b

Statement b claims that a primer must be prepared with a complementary sequence to part of the DNA of interest. This is true as primers provide the starting point for DNA synthesis.

Evaluate Statement c

Statement c states that repeated heating and cooling cycles allow the enzymes to act specifically and replaces helicase. This is true, as the heating step denatures the DNA, separating the strands without the need for helicase.

Evaluate Statement d

Statement d claims that each cycle of PCR doubles the amount of DNA of interest. This is also true as each cycle results in the replication of the DNA, doubling its quantity.

Identify the False Statement

After evaluating all statements, it is clear that statement a is false. Human DNA polymerase is not used in PCR.

Key concepts

DNA Amplification

DNA amplification is a central concept in the Polymerase Chain Reaction (PCR) process. It refers to the exponential increase of a specific DNA segment through repeated cycles of replication. The primary goal is to generate large quantities of a DNA sequence from a small initial sample.

During PCR, the DNA of interest is duplicated multiple times. Each cycle of PCR results in doubling the amount of the targeted DNA, leading to exponential growth.

This process is essential in various applications like genetic fingerprinting, cloning, and forensic analysis. Successfully amplifying DNA ensures that there is enough genetic material to study and analyze in detail.

• **Key Point**: Each PCR cycle doubles the amount of DNA, which is fundamental to amplification.
• **Importance**: Amplification allows for adequate DNA quantity even from minimal samples.

Taq Polymerase

Taq polymerase is a heat-resistant enzyme crucial for the replication phase of PCR. It is named after the bacterium Thermus aquaticus, from which it is extracted. Unlike human DNA polymerase, Taq polymerase can withstand the high temperatures required for DNA denaturation in PCR.

The enzyme synthesizes new DNA strands by adding nucleotides to the primed single-stranded DNA. This robustness to thermal cycling makes Taq polymerase indispensable for PCR.

• **Heat-Resistant**: It remains functional at high temperatures necessary for PCR.
• **Role**: Facilitates the synthesis of new DNA strands, allowing repeated cycles of denaturation, annealing, and extension.

PCR Cycle

The PCR cycle is a series of steps that result in the amplification of the DNA segment of interest. Each cycle includes three main stages: denaturation, annealing, and extension. This cycle is repeated multiple times to achieve exponential DNA amplification.

**Denaturation**: The double-stranded DNA is heated to around 94-98°C to separate it into two single strands.

**Annealing**: The temperature is lowered to 50-65°C to allow primers to bind or 'anneal' to the complementary sequences on the single-stranded DNA.

**Extension**: Taq polymerase extends the primers by adding nucleotides to synthesize new DNA strands, at about 72°C.

• **Denaturation**: Unzipping of the DNA strands due to high temperature.
• **Annealing**: Binding of primers to the DNA template.
• **Extension**: Addition of nucleotides to form new DNA strands.

Primer Annealing

Primer annealing is a critical step in the PCR process where the primers bind to specific sequences on the single-stranded DNA template. Primers are short, single-stranded sequences of DNA that are complementary to the target DNA region.

This binding occurs during the annealing phase when the temperature is lowered to facilitate the hydrogen bonding between the primer and the template.

The specificity of the primers ensures that only the target DNA segment is amplified.

• **Specificity**: Primers must match the complementary sequence of the DNA for accurate binding.
• **Binding Temperature**: Typically set between 50-65°C to allow hydrogen bonds to form between the primer and the template strand.

The correct annealing of primers is essential for the success of the PCR, as it sets the stage for subsequent DNA synthesis by Taq polymerase.