| Description |
The overall speed of a polymerase chain reaction (PCR) is determined by many factors including the time required for denaturation during each cycle. Indirect evidence suggests that denaturation during PCR is complete in less than a second. Therefore, our experimental observations of DNA denaturation were conducted with a stop-flow instrument having a ultrashort time resolution of 1.2 ms. Duplex denaturation under PCR conditions was monitored with a fluorescent dye using PCR products of 51, 100, 272, and 547 bp. The 100 bp product showed a t1/2 of 100 ms when the temperature was 2.4 C above the Tm. Rates increased rapidly at higher temperatures. However, rates did not correlate with the length of the PCR product; the longest (547 bp) product had 2 melting domains. Our results suggest that adequate denaturation time and temperature control are necessary refinements for successful high-speed PCR. |
