Figure 1

Dual labelling strategy for FACS analysis. (A) Schematic diagram of an emPCR sample labelled with random hexamers and SYBR Green in combination with a biotin-conjugated target-specific probe and streptavidin-conjugated Alexa647. The sample contains (I) non-fluorescent naked beads, (II) unspecific product-carrying beads displaying SYBR Green labelling (green ellipse) and (III) beads carrying amplicon target product displaying strong SYBR Green labelling and Alexa647 (purple star) labelling. (B) FACS analysis revealed two populations of DNA-covered SYBR Green fluorescent beads (arrows) in the sample (I) and a SYBR Green non-fluorescent population in both the sample and the negative control sample containing labelled naked beads (II). (C) An Alexa647 fluorescent bead population (purple brackets) with an amplified gene fragment on their surface is evident in the sample (I), while the negative control only showed the Alexa647 negative population that was also present in the sample (II). (D) FACS dotplot of the labelled sample demonstrates the distribution of SYBR Green signals, with an overlap at the higher intensities with beads that also carried the Alexa647 target-specific probe (purple dots). Beads in the square brackets were collected for sequencing by the Roche/454.