4.1.1 Overview

PCR (Polymerase Chain Reaction) amplification efficiency measures how effectively a DNA template is replicated in each cycle. In an ideal PCR reaction, the DNA amount doubles every cycle, corresponding to an amplification efficiency of 100%. However, in practice, amplification efficiency often falls short of this assumption due to various factors, including,

• Template Quality: Degraded or impure DNA can hinder the PCR process.
• Primer Design: Primers must specifically and efficiently bind to the target DNA, avoiding secondary structures or dimers.
• Reagent Concentrations: Optimal levels of dNTPs, MgCl2, and polymerase are essential for efficient amplification.
• Thermal Cycling Conditions: Proper annealing and extension temperatures and times are necessary for effective DNA synthesis.
• Inhibitors: Substances like ethanol, phenol, or excessive salts can inhibit the PCR reaction.

Amplification efficiency is often quantified by examining the exponential phase of the PCR reaction through quantitative PCR (qPCR).

Sparkling

Thus, studying PCR amplification efficiency at various rates can help us understanding how reads are generated and rendered in the sequencing library finally.