Doubling Throughput of a Real-Time PCR

Abstract

The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

Figure 1

(A) A typical data set with both amplification curves extracted from a 40-cycle PCR. The fluorescence amplitude recorded at the end of the extension (red) and denaturation (green) step are shown here. The normalized amplification curves are shown as inset. At the end of the PCR we performed the MCA. (B) The results of the MCA analysis showing DNA templates with two different melting temperatures, 78.6 °C and 84.3 °C.

Figure 2. Raw fluorescence data from LightCycler for experiments with different ratios between HPRT and GAPDH genes.

(A) Shows HPRT gene alone, (B) ratio 1:1, (C) ratio 1:2 and (D) ratio 1:10. We extracted both denaturation (green) and extension (red) curves from all graphs. No template control (NTC) expressed no amplification and its average fluorescence amplitude was 0.113 with a standard deviation of 0.002.

Figure 3. (left axis) Extracted normalized extension curves.

The curves’ amplitude was divided by its maximum amplitude and the slope was also subtracted for easier curve to curve comparison. (right axis) The normalized denaturation curves. The normalization was done in a similar fashion as for extension curves.

Figure 4

Extracted threshold cycles (C_T) for experiments with fixed concentration of either HPRT (A,B), GAPDH (C) or HA (D) genes and varying concentrations of GAPDH (A) HA (B) or HPRT (C,D). Each experiment was repeated three times. The varied concentration was calculated by subtracting the constant value of concentration (6.25 × 10⁻⁷ ng/μL) from the total DNA concentration. The red circles correspond to CT extracted from the denaturation curves while the blue squares from the extension curves.