Multiplex primer prediction software for divergent targets

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

These values indicate that the sequenced product was vaccinia DNA and not amplification from an exogenous nucleic acid source, e.g. host cell. Notably, our multiplex reaction did not produce a smear (lane 3, Figure 7 ) that would be indicative of nonspecific priming of either the target or exogenous host cellular nucleic acids. While the exact manufacturer's extraction protocol is proprietary, it is generally known that a standard sucrose gradient ultracentrifugation step is used to enrich for the viral capsids prior to viral nucleic acid extraction. However, to the best of our knowledge, no nuclease digestions are performed prior to viral capsid lysis. Furthermore, while the viral 'extract' contains a mixture of both viral DNA and cellular nucleic acids, the exact proportions of host cell and viral nucleic acids cannot be determined. Thus, our results indicate that a multiplex of 16 primers of 10 nt each amplifies only the specific predicted band from vaccinia.