Question

Which of the following is correct for the Primer Annealing Temperature in a PCR?
(A) Base composition and length of the template DNA
(B) Melting temperature of the primer
(C) Base composition and length of the primer
(D) Genomic DNA content of the plant tissue
Choose the correct answer from the options given below:

• (A) and (B) only.
• (A) and (C) only.
• (B) and (C) only.
• (C) and (D) only.

Hint:

A simple formula used to estimate the melting temperature ($T_m$) of short primers ($14-20\text{ bp}$) is the Wallace-Itakura formula:
\[ T_m = 2(A + T) + 4(G + C)\text{ }^\circ\text{C} \]
This highlights how $T_m$ depends directly on the primer's base composition.

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
The Polymerase Chain Reaction (PCR) is an in vitro technique used to amplify specific DNA sequences.
A PCR cycle consists of three thermal steps: denaturation, annealing, and extension.
During the annealing step, the reaction temperature is lowered to allow the oligonucleotide primers to bind specifically to their complementary target sequences on the single-stranded template DNA.

Step 2: Detailed Explanation:
Let us analyze the factors that determine the optimal annealing temperature ($T_a$):
The optimal annealing temperature is determined primarily by the melting temperature ($T_m$) of the primers, and is typically set $3-5\text{ }^\circ\text{C}$ below the $T_m$ of the primer with the lower melting temperature.
Thus, Statement (B) is correct.
The melting temperature ($T_m$) of a primer depends directly on its length and base composition (GC content).
Because G-C base pairs share three hydrogen bonds while A-T pairs share only two, primers with higher GC content require higher temperatures to melt and anneal.
Thus, Statement (C) is correct.
The overall size, concentration, or base composition of the large genomic template DNA does not determine the optimal annealing temperature of the short primers.
Thus, Statement (A) is incorrect.
The total genomic DNA content or mass extracted from the plant tissue does not influence the annealing temperature of the primers.
Thus, Statement (D) is incorrect.
Evaluating the statements shows that only (B) and (C) are correct.

Step 3: Final Answer:
Statements (B) and (C) are correct.
This corresponds to Option (C).