| Abstract
| - A novel method for multiplex TaqMan PCR in nanolitervolumes on a highly integrated silicon microchamberarray is described. Three different gene targets, relatedto β-actin, sex-determining region Y (SRY), and RhesusD (RhD) were amplified and detected simultaneously onthe same chip by using three different types of humangenomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternatedispensing of mineral oil-coated microchambers containing template and those without template. To confirm thespecificity of our system to β-actin, SRY, and RhD genes,we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The sampleswere cycled with the same sustaining temperatures as withthe microchamber array. Instead of the conventionalmethod of DNA quantification, counting the number ofthe fluorescence released microchambers in consequenceto TaqMan PCR was employed to our chip. This simplemethod of observing the end point signal had provided adynamic quantitative range. Stochastic amplification of0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highlysensitive response for simultaneous multiple-target detection, which is a promising step toward the developmentof a fully integrated device for the “lab-on-a-chip” DNAanalysis.
|
