The Polymerase Chain Reaction (PCR) is one of the most useful and generally applicable protocols in molecular biology and forms the basis of an increasing number of diagnostic tests and other assays. It would be highly desirable to be able to screen a single sample from an individual or location for the presence of many infectious agents simultaneously by PCR, i.e. to perform multiplex PCR. Presently, difficulties encountered when increasing the PCR multiplex depth include (a) the limited number of resolvable optical signatures available for optical multiplexing and (b) the substantial bioinformatic design issues related to minimizing the complex and deleterious interactions among primers, templates and amplicons which can lead to nonspecific amplification. We propose to address these impediments by carrying out Solid Phase PCR (SPPCR) directly on the surface of thermally stable, porous glass beads which have been encoded with Parallume optical encoding technology (www.parallume.com) and with one or both of the two primers covalently bonded to the surface of the bead. The large number of optical codes available from the rare earth-based Parallume encoding technology, combined with a reduction in primer-primer and primer-amplicon interactions by sequestering one or both of the primers onto the bead surface, will provide a PCR methodology with a multiplexing depth greater than any current technology. In Phase I we will evaluate the proposed multiplex PCR in terms of ease of use, sensitivity and selectivity in collaboration with the Chemical and Biological Countermeasures (CB) Division at Lawrence Livermore National Laboratory (LLNL). We will perform a Parallume bead-based 10-plex PCR using three signatures from Yersinia pestis (Plague), three from Bacillus thuringiensis Israeliensis and four from Saccharomyces cerevisiae. The ten forward primers will be covalently attached to beads with ten different optical codes and the beads added to a standard PCR mix containing the 10 reverse primers (which may be labeled) in solution. The PCR product will be quantitated by either (a) directly measuring the increasing fluorescent intensity on each bead from incorporation of a Cy3 (or phycoerythrin)-labeled reverse primer, (b) indirectly by hybridization of a complementary Cy3-labeled probe onto the bound amplicon after denaturing or (c) determination of the uptake of an intercalation dye like SYBR or Pico Green. The sensitivity and selectivity of Parallume encoded bead system for PCR will be evaluated by comparison of our bead results to a serial dilution series of corresponding Taqman assays performed at LLNL.
This innovation seeks to perform highly-multiplexed solid phase PCR with primers attached to a porous glass bead optically-encoded with the Parallume technology. The end result of this process will be an assay where an unknown sample (e.g., blood, soil, tissue, air) can be simultaneously analyzed for known biothreats or infectious diseases based on the detection of a known DNA fragment from those species. Segregating either the forward or both the forward and reverse primers on the large surface area of the porous beads prohibits their interaction with primers on other beads thereby diminishing the probability of nonspecific amplification products to occur from primer-primer interactions. Using this methodology, a multiplexed solid phase PCR reaction can be used in place of solution phase PCR where the high cost and tedious experimentation in developing highly specific primers lead to a long lag time between the initial development of a multi-target assay and the finished product. In addition to the current methods being solution-based, there is currently a high cost of the fluorescent probes used in the detection of the amplified product and a high cost to the method of detection. Both of these higher costs are mitigated by the use of Parallume-encoded beads and the MARS analysis system on which to run the PCR and analyze the post-PCR beads, respectively. ? ? ?
