Tentacle Probes: differentiation of difficult single-nucleotide polymorphisms and deletions by presence or absence of a signal in real-time PCR.
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, 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.
Satterfield
Brent C
BC
Harrington Department of Bioengineering, Arizona State University, Tempe, AZ 85287-9709, USA.
Kulesh
David A
DA
Norwood
David A
DA
Wasieloski
Leonard P
LP
Caplan
Michael R
MR
West
Jay A A
JA
D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics; D001409:Bacillus cereus / Q000145:classification; D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques; D027081:DNA Gyrase / Q000235:genetics*; D005189:False Positive Reactions; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D015010:Yersinia pestis / Q000145:classification* / Q000235:genetics; D015011:Yersinia pseudotuberculosis / Q000145:classification
D001426:Bacterial Proteins; D027081:DNA Gyrase