PCR efficiency — In later cycles, the amplification efficiency of PCR often declines primarily due to which combined reasons?

Introduction / Context:
While PCR is exponential in early cycles, yields eventually plateau. Understanding why the reaction slows helps optimize protocols, troubleshoot poor yields, and interpret quantitative data. Multiple factors converge to limit efficiency as the reaction proceeds.

Given Data / Assumptions:

• dNTPs and primers are finite and become limiting over time.
• Polymerase activity can decline; extension time may be insufficient as products lengthen.
• Accumulated amplicons can re-anneal to themselves or form secondary structures.

Concept / Approach:
The “plateau effect” reflects resource depletion (substrates like primers and dNTPs), enzyme performance limits (partial denaturation/inactivation, limited extension time), and product inhibition (amplicon-amplicon reannealing competing with primer binding). These factors collectively reduce the effective amplification per cycle, making “all of these” the correct integrative choice.

Step-by-Step Solution:

Verification / Alternative check:
qPCR curves show sigmoidal kinetics: early exponential phase gives way to a plateau despite further cycling, reflecting these constraints.

Why Other Options Are Wrong:

• Single-cause answers miss the multifactorial nature of the efficiency decline.

Common Pitfalls:
Attempting to “fix” plateau phase by merely adding more cycles; without addressing limiting factors, extra cycles add little product.

Final Answer:
all of these