DNA rehybridization during PCR: the 'Cot effect' and its consequences.
The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries.
Mathieu-Daudé
F
F
Sidney Kimmel Cancer Center, San Diego, CA 92121, USA.
Welsh
J
J
Vogt
T
T
McClelland
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA; D005188:False Negative Reactions; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016053:RNA, Protozoan / Q000032:analysis*; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D016053:RNA, Protozoan; D004247:DNA