Potential influence of the first PCR cycles in real-time comparative gene quantifications

Abstract

There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1-3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

“…PCR products of varying length are created during initial stages of the PCR and these products only influence reaction kinetics during the first few cycles [20]. Our work implies that template quality also impacts PCR efficiency only during these stages [21].…”

“…Nogva and Rudi observed that the Listeria monocytogenes 23S rRNA gene amplifies less efficiently than its hlyA gene and that restriction enzyme digests of input genomic DNA improve the amplification efficiencies of both genes. They concluded that amplification efficiency during early PCR cycles is less than the efficiency during the exponential phase [20]. Work in our lab adds template quality as a factor that impacts early cycle reaction efficiency [21].…”

Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

“…4). It has been reported that qPCR quantification bias between 2 genes (linear DNA) originated from the amplification efficiency of first PCR cycle [6]. In the theoretical model proposed by Nogva and Rudi [6], 3 types of amplification were occurring during the PCR process (Fig.…”

Detection and Measurement of Fungal Burden in a Guinea Pig Model of Invasive Pulmonary Aspergillosis by Novel Quantitative Nested Real-Time PCR Compared with Galactomannan and (1,3)-β- d -Glucan Detection

“…With our nested PCR assay, the first round of amplification with panfungal primers enriches the fungal DNA fraction and improves the fungal versus nonfungal DNA ratio. Specific oligonucleotides used in the second round can therefore easily find their targets, allowing for more-efficient PCRs (19). The preamplification step enables the detection of very low copy numbers in blood samples.…”