Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

Abstract

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

Figure 1

Principle of the proposed qPCR multiplexing method. (A) We prepared the PCR master mix containing two DNA templates, two sets of relevant forward and reverse primers, a FAM probe specific to one DNA template, and EvaGreen intercalating dye. (B) We performed the PCR and captured the F (green curve) during each cycle at the end of the denaturation and elongation steps, while the red curve represents the heater temperature. Contributions to the F at the denaturation step are only from the FAM probe (inset B1). F at the elongation step consists of both the FAM probe linked to Chr21 as well as the EvaGreen intercalating dye with both targets (inset B2). (C) All of the collected data during the PCR followed by the MCA were split (D) into two sets and plotted separately, which represents the denaturation amplification curve (red) and the elongation amplification curves in gradient colors from dark to light green for different cn₁₈. (E) We also plotted an MCA as well as the negative derivation of F showing the presence of two different amplicons in the PCR master mix.

Figure 2

Determination of the copy numbers of both targets based on calculation. (A) Values of C_tD and C_tE as a function of cn₂₁ with cn₁₈ as the parameter. The black lines with squares are three denaturation curves overlapping each other that have been calculated using eq 1. The blue curve with downward triangles, the green with upward triangles, and the red with circles are elongation curves calculated by eq 2. Each of them represents different values of cn₁₈. (B) ΔC_t values as a function of R_SET with cn₁₈ as the parameter that has been extracted from Figure 2A. All three curves overlap, which shows that it is the R_SET value determining the ΔC_t instead of an absolute value of cn₁₈.

Figure 3

C_t values extracted from the PCR with EvaGreen intercalating dye and the FAM probe. (A) PCR standard curves of the variable cn₂₁ values with three different cn₁₈ values as the parameter with the denaturation curves marked with circles and the elongation with squares. The cn₂₁ values could be determined in an entire range from the denaturation standard curves. The arrow pointed to the curved region where minor Chr21 clearly influences Chr18 amplification. The green line marked with triangles was formed by the curve fitting of an average value from all three denaturation curves. (B) Values of ΔC_t plotted as a function of R_SET showing the importance of the R_SET value and not the individual cn₂₁ or cn₁₈. (C) Equivalent of (A) with cn₁₈ as the variable and cn₂₁ as the parameter with elongation curves marked by squares and the denaturation by circles. (D) Equivalent of (B) with cn₁₈ as the variable and cn₂₁ as a parameter.

Figure 4

C_t values extracted from the PCR with FAM and VIC probes. (A) FAM channel with a cn₂₁ variable with cn₁₈ as the parameter showing the problems with the dual-channel amplification as the standard curves with different values of cn₁₈ should be collinear and form a single line regardless of the cn₁₈ value. (B) Same PCR mixtures using a VIC channel also showed the influence of cn₂₁ on cn₁₈ as the results should be independent of cn₂₁ since it was Chr18 with the VIC probe. (C) Implementation of the 2^–ΔΔCt method showing its shortcoming with the noncompensated data for fluorescence spillover. (D–F) Similar results for a mixture with the cn₁₈ value variable and the cn₂₁ as the parameter.