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

Sammendrag

There is an underlying assumption in real-time quantitative 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 focus on the first cycles in PCR in explaining the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (Type 1 to 3) dominating in the first cycles. Type 1 is the original target, Type 2 are undefined long products, while Type 3 is the product that accumulates during PCR. We evaluated the effects of the 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 amplicons. The cutting 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 consider the effect of the first few PCR cycles for the conclusions drawn