What is PCR Efficiency?
PCR efficiency is defined as the percentage of target molecules that are copied per PCR cycle. Imagine a reaction tube that initially contains 100 target molecules. After one round of PCR, it now contains 190 target molecules. The PCR efficiency of this reaction is 90%, because 90% of the target molecules were amplified.
In any reaction, the ideal PCR efficiency is 100%. This indicates amplification of all of the target molecules with each successive cycle. However, a PCR efficiency of 100% is not always attainable. A PCR efficiency ranging from 90% to 110% is considered acceptable.
Why is PCR Efficiency Important?
If the underlying goal of the reaction is to quantify the total amount of target molecules in a given sample (quantitative PCR), a high PCR efficiency value is essential, as it is a reflection upon the accuracy of the data obtained from the reaction.
Calculating PCR Efficiency
PCR efficiency is calculated from the standard curve of an assay. A standard curve is created by preparing 5 individual serial dilutions of a control DNA template, each diluted by a factor of ten, against which the unknown target template can be measured. Each serial dilution is used to perform a separate real-time reaction. The subsequent Cq values are plotted and the standard curve is generated by fitting a linear line to the data points. The PCR efficiency can then be determined from the slope of the standard curve.
Low PCR Efficiency
The efficiency of a reaction may be negatively affected by a combination of a wide variety of factors. Some of the most common factors include:
- Sample inhibition due to inhibitory compounds
- Non-optimal PCR primer and/or probe design
- Primers designed on a single nucleotide polymorphism (SNP) site
- Incomplete DNAse treatment (if applicable)
- Formation of primer dimers (PDs) or non-specific amplification
- Dynamic range of the standard curve is not large enough
- Incorrect sample dilutions
- Pipetting errors across the standards and/or samples