Kirk M. Ririe

Rapid Cycle DNA Amplification

DNA amplification requires temperature cycling of the sample. From the viewpoint of the sample, the only relevant characteristics of a temperature cycler are its speed and homogeneity. How fast the sample temperature can be changed largely determines the cycle time. How uniform the sample temperature is affects reproducibility. As cycle speed increases, it becomes harder to maintain homogeneous temperatures within and between samples. Standard commercial instrumentation usually completes 30 cycles (94, 55, 74°C) in about 2–4 hr. A new “high-performance” system requires about half as much time and is reported to run two temperature profiles (60, 94 °C) in a little over an hour (Haff et al., 1991). “Rapid cycle DNA amplification” as used here refers to completion of 30 cycles of amplification in 10–30 min.

Fluorescence Monitoring of Rapid Cycle PCR for Quantification

The polymerase chain reaction (PCR) benefits from rapid temperature cycling (Wittwer et al., 1994). In particular, rapid cycling appears to improve the quantitative PCR of rare transcripts (Tan and Weis, 1992). The glass capillaries used as sample containers for rapid cycling are natural cuvettes for fluorescence analysis. Fluorometric monitoring of PCR has been reported with double-stranded DNA (dsDNA) dyes (Higuchi et al., 1992; Higuchi et al., 1993; Ishiguro et al., 1995; Wittwer et al., 1997a) and sequence-specific probes (Lee et al., 1993; Livak et al., 1995; Wittwer et al., 1997a). We have integrated a fluorimeter with a rapid temperature cycler for fluorescence monitoring during amplification (Wittwer et al., 1997b). Both cycle-by-cycle fluorescence monitoring and continuous (within cycle) monitoring offer unique quantitative information.