Mar 26, 2015

Accurate Isothermal Amplification Reaction Rate Determination Using High-Frequency Sampling

BioRxiv : the Preprint Server for Biology
Thomas P Beals

Abstract

Background Nucleic acids quantification by amplification is currently done primarily by real-time amplification for relative quantification, or by statistical inference from replicated endpoint assays for absolute quantification. The polymerase chain reaction (PCR) has been the dominant amplification technology, although alternative isothermal technologies have been described. Theoretical analysis of amplification kinetics and of amplification data interpretation have almost exclusively considered the PCR. Results Real-time measurements of isothermal amplification reactions can be made continuously, in contrast to the discrete per-cycle measurements of real-time PCR. Isothermal ramified rolling circle amplification (RAM) reactions were measured at frequent intervals, and amplification data subsets were fitted to an exponential amplification model. Signal-change-over-time slopes and time-zero signal intercepts were derived from the chosen subset data. Slope measurements were sufficient to determine a reaction rate (the isothermal equivalent of PCR efficiency) for each reaction. Analysis of slope and intercept together suggest that amplification reactions that were initiated from a single target molecule can be distinguished fro...Continue Reading

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Mentioned in this Paper

Real-Time Polymerase Chain Reaction
Determination Aspects
Nucleic Acid Amplification Tests
DNA Amplification Techniques
Theoretical Study
Gene Amplification Technique
Time Measurements
Monitoring - Action
Analysis
Nucleic Acids

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