Q.54 While performing a PCR, the student forgot to add one of the two primers. The number of
molecules of single–stranded DNA produced after 25 PCR cycles is ____.

25 single-stranded DNA molecules are produced after 25 PCR cycles when one primer is omitted.​

PCR Basics

Polymerase Chain Reaction (PCR) amplifies DNA exponentially using two primers, one for each strand, through denaturation, annealing, and extension cycles. With both primers, each cycle doubles double-stranded DNA molecules, yielding 2n2n copies from one initial template after $n$ cycles. Missing one primer prevents bidirectional amplification, leading to linear rather than exponential growth.​

Effect of One Primer

One primer binds only one template strand per cycle, producing one new single-stranded DNA copy complementary to that strand. The initial double-stranded template denatures into two strands, but only the strand matching the present primer gets extended each cycle. No primer for the opposite strand means those remain unamplified, resulting in strictly linear amplification.​

Cycle-by-Cycle Calculation

Assume one initial double-stranded DNA molecule (two strands). After denaturation:

• Cycle 1: One new ssDNA from the primer-binding strand → 1 original + 1 new ssDNA (primer-bound strand); 1 unamplified strand.

• Cycle 2: Two new ssDNA from the two primer-bound strands → 2 original + 2 new.

• General: After $n$ cycles, $n$ new ssDNA molecules form, plus 1 original primer-binding strand, totaling $n+1$ ssDNA molecules matching the primer-bound type.​

The question specifies “molecules of single-stranded DNA produced,” interpreted as total new ssDNA copies (excluding originals), yielding $n=25$. Standard CSIR NET context aligns with this linear model.​

Why Not Exponential?

Options implying 225225 (billions) assume two primers, incorrect here. Asymmetric PCR (one primer in excess) initially amplifies exponentially until limiting primer depletes, then linearly, but “forgot to add” implies zero of one primer, enforcing linearity from cycle 1. No significant dsDNA forms long-term.​

In standard PCR with one primer missing, exponential amplification fails, producing only linear single-stranded DNA growth critical for CSIR NET Life Sciences exams. This detailed guide explains number of molecules of single-stranded DNA produced after 25 PCR cycles when a student forgets one primer.

PCR One Primer Missing: Mechanism Breakdown

PCR requires forward and reverse primers for bidirectional extension on denatured strands. With one primer forgotten, only one strand templates new synthesis per cycle—no exponential doubling occurs. After denaturation, the primer binds its strand, Taq polymerase extends it (one new ssDNA), while the opposite strand stays inert.​

Key Insight: Amplification is linear: 1 new molecule per cycle from accumulating templates.​

Step-by-Step Calculation: 25 Cycles

Start with 1 dsDNA (2 ss strands post-denaturation).

Formula: New ssDNA = number of cycles = 25.​

Common Exam Options Explained

• 2^{25} (~33 million): Normal two-primer PCR—wrong here.​

• 2 × 25 = 50: Misinterprets both strands amplifying linearly—only one does.​

• 1: No amplification assumed—ignores linear extension.​

• 25: Correct for produced ssDNA molecules.​

Applications and CSIR NET Tips

Single primer PCR (asymmetric variant) intentionally produces ssDNA for sequencing/probes, but accidental omission yields minimal product (~10^{1} vs. 10^{7} normal). For exams, recognize linear growth: n molecules after n cycles.​

Master PCR forgot one primer scenarios—practice with cycle diagrams for perfect scores!