1. xTAG liquid-phase microarray.
xTAG liquid-phase microarray. Target sequences (blue and green) are amplified using multiplex PCR. Following amplification, a second set of target-specific primers containing “universal tag sequences” (orange and red boxes) unique to each target primer are used for a primer extension reaction. During primer extension, a biotin label is also incorporated into the amplicon. Labeled amplicons are then incubated with polystyrene microbeads. Microbeads are uniquely colored, allowing differentiation of up to 100 different types of microbeads by the analyzer. Each color bead is also coated with a single-strand nucleic acid probe complementary to one of the universal tag sequences (antitag). Amplicons labeled with universal tag sequences will hybridize to the microbeads containing the antitag. Additionally, a streptavidin-fluorophore conjugate (green star) is added and hybridizes to biotin-labeled amplicons immobilized on the beads. Following hybridization steps, beads are analyzed using a cell sorter equipped with two lasers. The first detects the presence of the fluorophore conjugated to biotin, indicating the presence of an amplicon bound to a specific microbead. The second laser interrogates the bead to determine which dye is present, thereby identifying the specific target amplicon present. The center bead in step 5 lacks amplicon and thus would be negative for the biotin-fluorophore signal. This bead would not be analyzed by the second laser.
Blake W. Buchan, and Nathan A. Ledeboer Clin. Microbiol. Rev. 2014; doi: /CMR