Multiplex primer prediction software for divergent targets

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

This multiplex was compared against the human genome, predicting 233 amplicons between 50 and 1000 bp from the Poxviridae 16-plex. For empirical tests against background human nucleic acids, we followed the same reaction conditions as above. In these tests, vaccinia DNA was held at a constant concentration of 2.7 pg (1.3 Â 10 4 copies), and serial dilutions of human genomic DNA (Novagen, Madison, WI, USA), were added to the vaccinia PCR reaction mix, starting at 2.7 pg and titrating down over 4 orders of magnitude to 2.7 Â 10 -4 pg. These experiments were performed using mass ratios of vaccinia:human DNA at 1 : 1, 10 : 1, 100 : 1, 1000 : 1 and 10000 : 1. These correspond to copy number ratios of vaccinia:human genomes of 1. All PCR experiments were analyzed on 3% agarose TBE gels containing ethidum bromide that were purchased from Bio-Rad (Hercules, CA, USA). Blue juice TM 10Â loading dye was purchased from Invitrogen (Carlsbad, CA, USA) and diluted to 2Â before use. A 50-bp DNA ladder was purchased from Novagen (Madison, WI, USA). For analysis, 15 ml from each separate 25 ml PCR reaction were combined with 2 ml of of loading dye and 15 ml of the loading-dye/product mixture was loaded per well and electrophoresed for 1 h 40 min at 85 V.