Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

Abstract

False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation. Tentacle Probes, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan-minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve. With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred. The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.