Parallel-processing continuous-flow device for optimization-free polymerase chain reaction

摘要

A parallel-processing four-station polymerase chain reaction (PCR) device has been developed, which performs continuous-flow PCR without optimization of the annealing temperature. Since the annealing temperature of each station can be controlled independently, the device covers an annealing temperature range of 50-68 A degrees C, which is wide enough to perform PCR for any DNA fragment regardless of its optimum annealing condition. This arrangement lets us continuously obtain an amplified amount of a DNA fragment at least from one of the stations. The device consists of four identical cylindrical stations (diameter 20 mm, height 55 mm). A polytetrafluoroethylene capillary reactor (length 2 m, I.D. 100 mu m, O.D. 400 mu m) is wound helically up around each station. The whole assembly is designed to minimize the number of heating blocks (for providing temperatures of denaturation, annealing, and extension) to be seven and to shape a compact cube (height 55 mm, base 60 mm x 60 mm). The reproducibility for continuous-flow PCR is reasonably high (run-to-run and station-to-station relative standard deviation of their amplification is lower than 6 % and about 4 %, respectively). Performance on the optimization-free DNA amplification has been evaluated with four DNA samples with different annealing conditions and product sizes (323, 608, 828, and 1101 bp), which has demonstrated that in all cases, PCR is successful at least on one station. In addition, three DNA fragments with different lengths (323, 1101, and 2836 bp) have been successfully amplified in a segmented-flow mode without the carry-over contamination between segments. This result suggests that this device could serve as the PCR module of a continuous-flow high-throughput on-line total DNA analysis system integrating all necessary modules from cell lysis/DNA extraction to PCR product analysis.