A real-time PCR analyzer compatible with high-throughput automated processing of 2-/spl mu/L reactions in glass capillaries

摘要

This paper describes the development of a real-time polymerase chain reaction testbed for the analysis of up to 48 1- to 2-/spl mu/L reactions in glass capillaries. It is suitable for high-throughput experimentation when used as a downstream module for ACAPELLA-5K, an automated, high-precision, fluid-handling system developed in our laboratory over the past five years. The testbed features a laser-induced fluorescence scanner with high-sensitivity photomultiplier tubes for the detection of three spectral wavelengths. The scanner has a detection threshold of 1-nM fluorescein in a 1-/spl mu/L detection volume. Peltier-effect thermoelectric elements are used to control thermal cycles with a slew rate of 3/spl deg/C/s, and a set point tracking accuracy of /spl plusmn/0.5/spl deg/C. Analyzer design and performance are presented. Experimental results show a successful detection of 3 copies//spl mu/L of the albumin gene in serial dilutions of human genomic deoxyribonucleic-acid. Detection levels down to 4 copies//spl mu/L of the t(14;18) translocation associated with the cancer follicular lymphoma, was demonstrated in RL-7 cells. Note to Practitioners-Real-time polymerase chain reaction is a thermally driven in-vitro replication of specific segments of DNA, with simultaneous monitoring or reaction product by means of fluorescence detection. This process is important in biochemical analysis for the detection and/or estimation of gene concentration in DNA samples. By increasing the concentration of an initially undetectable gene and monitoring the kinetic history of the reaction, an estimate of the initial concentration can be computed. Although there exists a plethora of commercial real-time PCR instruments, none of these instruments are suitable for low-volume (single-digit microliters) reagents and high-throughput processing while preserving a high degree of fidelity to obtain reliable, repeatable results, and versatility (support for different fluorogenic probes, different operating modes, etc.). The A5K-automated fluid handling and sample preparation system addresses the high-throughput reaction preparation in small-volume glass capillaries. The testbed presented here is a downstream processing module for A5K. To address the challenges posed by the use of- low-volume, elongated reaction vessels, we employ a scanning optical system with a novel inline illumination method to address up to 48 capillaries. We empirically demonstrate that the system's performance is comparable to that of existing commercial real-time PCR platforms, and verify its repeatability and versatility. Future work will address the development of a more compact instrument that can be directly integrated with A5K.