Biotin-containing protein as a cause of false positive clones in gene probing with streptavidin/biotin

Using a streptavidin/biotin labeling technique, we unintentionally cloned a gene encoding a biotin carboxyl carrier protein, a subunit of biotin-dependent enzymes, from a genomic library of Streptococcus mutans strain UT-041. In colony lifts, the clone reacted positively to the streptavidin-containing detection system but could not be detected in Southern blot analysis. The amino acid sequence of the gene product, deduced from its nucleotide sequence, demonstrated all the features common to biotin carboxyl carrier proteins from other bacteria, indicating that the biotin carboxyl carrier protein in the clone had produced a "false-positive" (DNA probe-independent) reaction by binding to the streptovidin. To circumvent this problem with the detection system in gene probing in the future, we recommend that all positive clones be screened by direct incubation with streptavidin-alkaline phosphatase (SA-AP) in the absence of biotin-labeled probe DNA. Clones binding to SA-AP would be considered false positives.