A DNA biochip for on-the-spot multiplexed pathogen identification

Yeung, Siu-Wai; Lee, Thomas Ming-Hung; Cai, Hong; Hsing, I-Ming

The assay procedure used in this work is schematically represented in Figure 4 . It involves three main steps: sample preparation, target DNA amplification, and product detection, all performed within the same microchamber. Intact cells are first broken down by applying a high temperature (90 C, controlled by the on-chip heater and temperature sensor) to free the genomic DNA. To remove all the interfering substances (e.g. cell debris and protein) that may affect the subsequent DNA amplification process, magnetic particles are used to isolate the specific genomes. Biotinylated genome capture probes for the two model species are mixed with the intact cells before injecting into the microchamber. When the temperature is lowered to 50 C after the thermal lysis step, these probes hybridize to their complementary target genomes. These probeÀgenome hybrids are then isolated by the addition of the avidin-coated magnetic particles, followed by thorough washing. It is worth noting that the magnetic particles are pretreated with a small amount of the genome capture probes to minimize nonspecific adsorption of the interfering substances and other genomic DNAs. Subsequently, with the genomes captured on the magnetic particles serving as the template, asymmetric PCR is conducted to generate single-stranded rich target amplicons.