Quantitative PCR: Procedures and precisions |
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Authors: | Jerry Nedelman Patrick Heagerty Chip Lawrence |
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Institution: | (1) Wadsworth Center for Laboratories and Research, University at Albany School of Public Health and New York State Department of Health, P.O. Box 509, 12201-0509 Albany, NY, USA |
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Abstract: | We develop a multitype branching-process model for the Polymerase Chain Reaction (PCR). We apply the model to a comparison
of three methods for estimating the initial number of molecules of target present in a PCR. These three methods are: one which
uses a coamplified, internal control; one which uses an external control series; and one which uses simple extrapolation of
log outputvs time (no control). We identify assumptions for each method which permit mathematical analysis of bias and precision. All
three methods perform well if: (1) replication efficiencies are stable among reactions; (2) other method-specific conditions
on efficiencies are met; and (3) product accumulates exponentially throughout the range where it is observed. When replication
efficiencies vary among reactions but other optimal conditions for each method hold, the no-control and external-control methods
lose precision relative to the internal control method, but they may still perform satisfactorily for many applications. The
internal control method continues to perform well even if accumulation of product plateaus. This method depends, however,
on a condition we call equivalence of replication efficiencies, the attainability of which in practice remains to be proven. |
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