Suitability of SP6 RNA polymerase transcripts for in vitro viral diagnosis.

To the Editor: Application of DNA or RNA probes to microbiology and virology requires probes free of vector sequences, particularly vectors of bacterial origin, such as pBR322. Incomplete purification of inserts from vector sequences can produce a falsely positive result when nucleic acid probes are applied to tissue or body fluids that may be containmated with bacteria. A plasrnid vector such as pBR322 provides a convenient system for production of large quantities of probe, but repeated preparative electrophoresis on agarose gel is required for it to be adequately pure. We find that large inserts ( 10 kb) can be prepared so pure that up to 10 ng of pBR322 will not be detected in a dot blot assay, but smaller inserts (-4-6 kb) that are similar in size to pBR322 cannot be purified nearly as well. As little as 100 pg of pBR322 can be detected with inserts 4.9kb in size, even after three preparative-electrophoretic steps. We anticipated that if we used RNA