A PCR curve, in real-time Polymerase Chain Reaction (qPCR), visually represents DNA amplification throughout a reaction. This graphical display plots the fluorescent signal detected against the number of amplification cycles. Its purpose is to monitor the accumulation of newly synthesized DNA in real time. The curve’s shape and characteristics offer insights into the presence and initial quantity of target DNA in a sample.
How Real-Time PCR Generates the Curve
Real-time PCR generates the curve by continuously measuring the fluorescent signal produced as DNA amplifies. The reaction mixture includes fluorescent dyes or probes that bind to or are cleaved by newly synthesized DNA. As DNA doubles with each cycle, more dye molecules bind or more probes are cleaved, increasing fluorescence. The qPCR instrument detects this fluorescence after each amplification cycle.
The instrument then plots these fluorescence readings against the cycle number, creating the amplification curve. Early in the reaction, the fluorescence signal is low. As DNA amplification progresses, the signal rises above the background, reflecting the increasing amount of amplified DNA. Each point on the curve represents the total accumulated fluorescence.
Understanding the Phases of a PCR Curve
A PCR amplification curve displays distinct phases. The initial segment is the baseline or lag phase, where the fluorescent signal remains low. During this phase, DNA amplification occurs, but the product amount is too small for detection. This period accounts for the first few cycles.
Following the baseline, the curve enters the exponential phase, also called the log-linear phase. Here, amplified DNA doubles every cycle, leading to a rapid increase in fluorescent signal. This phase has the highest reaction efficiency, and the product amount is directly proportional to the initial target DNA. This part of the curve is used for quantitative analysis.
Finally, the reaction transitions into the plateau phase, where the amplification curve flattens. This occurs because reaction components become depleted or degraded. The accumulation of inhibitors or competing side reactions can also contribute to the plateau, causing amplification to slow and eventually cease. The maximum fluorescence signal at plateau does not directly correlate with the initial DNA quantity.
The Cycle Threshold Value
The Cycle threshold (Ct) value represents a point on the amplification curve used for quantifying initial DNA amounts. It is the cycle number at which the fluorescent signal crosses a predetermined threshold level, set above background noise. This threshold is placed in the early part of the exponential phase, where amplification efficiency is stable and reproducible. The Ct value is inversely related to the starting quantity of target DNA.
A lower Ct value indicates the fluorescent signal reached the threshold in fewer cycles, implying a greater initial amount of DNA template. Conversely, a higher Ct value suggests more cycles were required to reach the threshold, indicating a smaller initial quantity of DNA. For instance, a sample with a Ct of 15 contains significantly more starting material than a sample with a Ct of 25. This inverse relationship makes the Ct value a direct measure for relative or absolute quantification of nucleic acids.
Interpreting Common Curve Variations
A “good” amplification curve exhibits a sigmoidal shape, starting with a flat baseline, transitioning smoothly into a steep exponential phase, and then leveling off in the plateau phase. This shape indicates efficient amplification and reliable detection of target DNA. The slope of the exponential phase indicates the reaction’s amplification efficiency.
Variations from this ideal shape provide clues about the reaction’s performance or sample quality. A flat line, with no significant fluorescence increase, indicates a lack of amplification. This could be due to absent target DNA, a failed reaction, or issues with primers or reagents. A late-onset curve (high Ct value) or a shallow exponential phase may suggest a low initial concentration of target DNA, or PCR inhibitors that reduce amplification efficiency.
Sometimes, a curve might show a high or erratic baseline, making it difficult to determine the Ct value. This can be caused by excessive background fluorescence or instrument noise, potentially compromising quantification reliability. Observing the shape and characteristics of the amplification curve is a step in validating real-time PCR results.