THEORETICAL UNCERTAINTY OF MEASUREMENTS USING QUANTITATIVE POLYMERASECHAIN-REACTION

Current quantitative polymerase chain reaction (PCR) protocols are onl y indicative of the quantity of a target sequence relative to a standa rd, because no means of estimating the amplification rate is yet avail able. The variability of PCR performed on isolated cells has already b een reported by several authors, but it could not be extensively studi ed, because of lack of a system for doing kinetic data acquisition and of statistical methods suitable for analyzing this type of data. We u sed the branching process theory to simulate and analyze quantitative kinetic PCR data. We computed the probability distribution of the offs pring of a single molecule. We demonstrated that the rate of amplifica tion has a severe influence on the shape of this distribution. For hig h values of the amplification rate, the distribution has several maxim a of probability. A single amplification trajectory is used to estimat e the initial copy number of the target sequence as well as its confid ence interval, provided that the amplification is done over more than 20 cycles. The consequence of possible molecular fluctuations in the e arly stage of amplification is that small copy numbers result in relat ively larger intervals than large initial copy numbers. The confidence interval amplitude is the theoretical uncertainty of measurements usi ng quantitative PCR. We expect these results to be applicable to the d ata produced by the next generation of thermocyclers for quantitative applications.