Gene Quantification
Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur a
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Fram;ois Ferre The Immune Response Corporation
Editorial Advisory Board Bruce K. Patterson Northwestern University
Kary B. Mullis Vyrex Inc.
Philip L. Feigner Vical Inc. William N. Drohan American Red Cross
Michael 1. Heller Nanogen Inc.
Phillips Kuhl Cambridge Healthtech Institute
Michael Karin University of California at San Diego
Robert E. Sobol Sidney Kimmel Cancer Center
Willem P. C. Stemmer Maxygen Inc.
Forthcoming volumes in the series Rational Therapeutic Target in Angiogenesis J.M. Pluda and W.W. Li, editors Techniques in Localization of Gene Expression Bruce K. Patterson, editor The Biology of p53 Michael I. Sherman and Jack A. Roth, editors Molecular Genetic Profiling: Applications to Diagnostics and Disease Management Lance Fors, editor
Gene Quantification Fran1000). It will then be considered that:
(2)
Even in this restricted perspective, the estimation of the amplification rate could be extremely valuable. Since kinetic data have not been previously available, rigorous estimation of the amplification rate was difficult, so
Statistical Estimations of PCR Amplification Rates
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methods have been developed to bypass this step in the analysis of amplification data. Most of these techniques rely on standard sequences introduced in known quantities into the experimental design. Assuming that the standard and the target sequence (the one that must be quantified) have identical amplification rates, one can determine the initial copy number of the target, Nn,T' from the initial quantity of the standard, No,s, and the measurements of the amount of two amplified sequences, No,s and Nn,n without any direct computation of the amplification rate. The basis of this approach is the next relation, which can easily derived from Equation (2):
Given the hypothesis that ms = mn the ratio of the standard molecule number over the number of target molecules remains constant after any number of amplification cycles. However, violations of this hypothesis will result in a significant evolution of the ratio over time, changing as (mSmT-1y. For instance, when ms = 1.9, m T = 1.8, and n = 25, then there is a 3.86-fold difference between Nn,sINn,T and No,sINo,T' Factors causing different amplification rates for the standard and the target are probably more numerous than factors causing exactly the same amplification rates. Disparity may arise from minor differences in the sequences, from tube-to-tube differences, from sample- to-sample differences, and so on. Since the methodology based on ratios arose at a time when the amplification rates were difficult to measure, it is likely that violations of the hypothesis would not have been detected and taken into account, and quantitative estimations would then have been contaminated with a systematic error. When Q-PCR experiments are conducted for relative quantification purposes such as the comparison of the quantities of two molecules-a common situation in gene expression and mRNA quantification experiments - the same argument applies. Quantitative differences betwe
