ABSTRACT
Dispenser ................................................................................................... 35 3.5 Thermal Cycling and Detection of Fluorescence...................................... 35 3.6 On-Chip Quantitation of the Initial DNA Concentration ......................... 35 3.7 Specific Sequence Detection from a Single Human Chromosome-1
via Nanoliter-Volume PCR ........................................................................ 38 3.8 Conclusion ................................................................................................. 40 Acknowledgment ................................................................................................ 41 References ........................................................................................................... 41
The miniaturization of analytical devices by the technique of semiconductor microfabrication is attracting many scientists for its applications in medical and bioanalytical fields, such as genetic analysis [1], clinical diagnostics [2], drug screening [3], and environmental monitoring [4]. The potential advantages of miniaturized analytical devices include reduced consumption of sample volume, high sensitivity, shorter analysis time, portability that allows on-site monitoring,
32 Chromosome Nanoscience and Technology
and disposability [5]. In particular, the sensitivity of detection of amplified DNA in a trace volume polymerase chain reaction (PCR), when compared with the normal scale of reaction, was expected to be substantially higher. If the same amount of DNA were amplified from the same copy template, DNA concentration per unit volume would be higher in the trace volume. Miniaturized onchip DNA amplification devices have also been reported in the last decade by several investigators [6-9]. However, there are significant problems associated with PCR amplification on chips; for example, cross-talk contamination between chambers and rapid drying of the trace volume PCR solution at nanoscale volumes. Therefore, in order to resolve these problems, we have developed a new method employing an oil layer on the microchamber instead of glass or plastic film covers. The sample DNA mixture to be amplified was introduced into each microchamber of the array, through an oil layer, using a nanoliter dispensing system [10,11]. The feasibility of our microchamber array was further improved by the use of TaqMan PCR [12]. The amplified DNA was detected by a microarray scanner using TaqMan fluorescence chemistry. The quantitation of the initial DNA concentration present in a microchamber was achieved from 0 to 12 copies per chamber, not only by monitoring the real-time fluorescence intensity but also by observing the end-point fluorescence signal [13]. Additionally, targetspecific DNA amplification from a single chromosome was also attained using our microchamber [14].
