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중합효소 연쇄반응을 위한 연속흐름형 미세유체시스템의 개발

Development of Continuous-Flow Microfluidic Systems for Polymerase Chain Reaction

초록/요약

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 50–68oC 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 µm, O.D.: 400 µm) 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 × 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 bp, 608 bp, 828 bp, and 1,101 bp), which has demonstrated that in all cases, PCR is successful at least on one station. In addition, four DNA fragments with different lengths (323 bp, 1,101 bp, and 2,836 bp) have been successfully amplified in a segmented-flow mode without the carry-over contamination between segments. The 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. A continuous-flow real-time PCR system capable of rapid quantification of various DNA samples by integrating a laser-induced fluorescence (LIF) detection system for the online monitoring of PCR products has been developed. The thermal cycler (diameter: 30 mm, height: 104 mm) consists of a thermal insulating plastic core with a triangle-like cross section and three copper blocks having circular arrangement around the core. To give sufficient reaction time for various lengths of the amplified DNA, the portion of the extension block is doubled, and thus the relative residence time in each copper block has the ratio of 1:1:2. A high purity perfluoroalkoxy alkane capillary reactor (length: 6 m, I.D.: 100 µm, O.D.: 400 µm) is wound helically up around the thermal cycler. The 10-mm gap between an annealing and extension blocks provides a detection window for measuring fluorescence signals from the sample. The fluorescence signal from each cycle can be obtained by only one-time scanning with the laser beam along the length of the thermal cycler. Performance on the continuous-flow real-time PCR system has been demonstrated by amplifying DNA samples, human β-actin (amplification efficiency= 101%, standard curve y = 25.1 − 3.30x, R2 = 0.992, n = 3) and bcr-abl gene (amplification efficiency = 94.2%, y = 48.5 − 3.47x, R2 = 0.997, n=3). The amplification results indicate that our device shows similar performance as that of commercial real-time PCR machines (amplification efficiency = 101%, R2 = 0.999).

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목차

Abstract
Contents
List of Figures
List of Tables

Chapter 1. Introduction
1.1.Polymeras Chain Reaction (PCR)
1.1.1. Components
1.1.2. Process
1.2. Miniatyrized PCR Devices
1.2.1. Srationary PCR
1.2.2. Continuous-Flow PCR
1.3. References

Chapter 2. Parallel-Processing Continuous-Flow System for Optimization-Free PCR
2.1. Introduction
2.1.1. PCR Optimization
2.1.1.2. Primer Design
2.1.1.2. Annealing Tempaerature
2.2. Experimental Section
2.2.1. Chemicals and Reagents
2.2.2. Construction of the Continuous-Flow Multi-Station Thermal Cycler
2.2.3. Multi-Station PCR
2.2.4. Segmented-Flow PCR
2.3. Results and Discussion
2.3.1.Construction of the Continuous-Flow Multi-Station Thermal Cycler
2.3.1.1. Design
2.3.1.2. Temperature Profile
2.3.2. Multi-Station PCR
2.3.3. Segmented-Flow PCR
2.4. Conclusions
2.5. References

Chapter 3. Continuous-Flow Microfluidic System for Real-Time PCR
3.1. Introduction
3.1.1. Real-Time PCR
3.1.2. Real-Time PCR vs. Conventional PCR
3.1.3. Detection Chemistry
3.1.3.1. SYBR Green Ⅰ
3.1.3.2. TaqMan Probe
3.2. Experimental Section
3.2.1. Chemicals and Reagents
3.2.2. Construction of Continuous-Flow Real-Time PCR System
3.2.2.1. Thermal Cycler
3.2.2.2. LIF Detection for Real-Time Monitoring of DNA Amplification
3.2.3. Continuous-Flow Real-Time PCR
3.2.4. Data Processing
3.3. Results and Discussion
3.3.1. Construction of Continuous-Flow Thermal Cycler
3.3.2. Continuous-Flow Real-Time PCR
3.4. Conclusions
3.5. References
Summary (in Korean)

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