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Clinical Chemistry 53: 2042-2050, 2007. First published October 11, 2007; 10.1373/clinchem.2007.091488
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(Clinical Chemistry. 2007;53:2042-2050.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Brent C. Satterfield1,2, David A. Kulesh3, David A. Norwood3, Leonard P. Wasieloski, Jr3, Michael R. Caplan1,a and Jay A.A. West2

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

aAddress correspondence to this author at: Arizona State University, PO Box 879709, Tempe, AZ 85287-9709. Fax 480-727-7624; 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 ProbesTM, 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.






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