Received on May 4, 2007
Accepted on September 17, 2007

Molecular Diagnostics and Genetics

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

¹ Harrington Department of Bioengineering, Arizona State University, Tempe, AZ, and Arcxis Biotechnologies, Pleasanton, CA
² United States Army Medical Research Institute of Infectious Diseases, Frederick, MD
³ Harrington Department of Bioengineering, Arizona State University, Tempe, AZ
⁴ Arcxis Biotechnologies, Pleasanton, CA

^* To whom correspondence should be addressed. E-mail: Michael.Caplan{at}asu.edu.

Background: 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.

Methods: Tentacle Probes^TM, 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.

Results: 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.

Conclusions: 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.