20. A polymerase chain reaction (PCR) was set up with the following reagents: DNA template, Taq
polymerase, buffer, dNTPs, and Mg2+. Which one of the following is missing in the reaction mixture?
(a) Helicase (b) Single-stranded binding proteins
(c) Primers (d) Reverse transcriptase
Missing Component in Reaction Mixture Explained
PCR (Polymerase Chain Reaction) amplifies specific DNA segments efficiently, but the setup requires precise reagents. In a mix with DNA template, Taq polymerase, buffer, dNTPs, and Mg²⁺, one key element is absent, making amplification impossible.
Correct Answer
Primers are missing from the PCR reaction mixture. These short, synthetic oligonucleotides (typically 18-25 nucleotides) are essential to provide a starting point for Taq polymerase, defining the exact DNA region to amplify by annealing to complementary sequences on the template strands.
Option Breakdown
PCR mimics DNA replication in vitro through denaturation, annealing, and extension cycles, but lacks natural cellular machinery—only engineered components suffice.
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(a) Helicase: This enzyme unwinds double-stranded DNA in vivo replication by breaking hydrogen bonds. PCR achieves strand separation via high heat (94-98°C), eliminating the need for helicase.
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(b) Single-stranded binding proteins (SSBs): SSBs stabilize unwound single strands during cellular replication to prevent reannealing. Thermal cycling in PCR keeps strands apart without SSBs.
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(c) Primers: Correct choice. Without forward and reverse primers flanking the target sequence, Taq polymerase cannot initiate synthesis, as it requires a 3′-OH primer terminus.
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(d) Reverse transcriptase: Used in RT-PCR for RNA templates, converting RNA to cDNA first. Standard PCR targets DNA, so it’s unnecessary here.
PCR Reaction Components
A complete standard PCR mix includes:
| Component | Role |
|---|---|
| DNA template | Target sequence to amplify |
| Taq polymerase | Thermostable enzyme for DNA synthesis |
| Buffer | Maintains optimal pH and ionic conditions |
| dNTPs | Building blocks (dATP, dCTP, dGTP, dTTP) |
| Mg²⁺ | Cofactor for polymerase activity |
| Primers | Initiate synthesis (missing here) |
This table highlights why primers are indispensable for specificity and efficiency.
Why Primers Matter in PCR
Primers ensure exponential amplification over 20-40 cycles, yielding billions of copies from nanograms of starting DNA. Mismatched or absent primers lead to no product, underscoring their role in techniques like diagnostics, forensics, and cloning.
