Surpassing specificity limits of nucleic acid probes via cooperativity

Brent C. Satterfield, Matt Bartosiewicz, Jay A A West, Michael Caplan

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Original languageEnglish (US)
Pages (from-to)359-367
Number of pages9
JournalJournal of Molecular Diagnostics
Volume12
Issue number3
DOIs
StatePublished - May 1 2010

Fingerprint

Nucleic Acid Probes
Freezing
Single Nucleotide Polymorphism
Temperature
Molecular Pathology
DNA Probes
Diagnostic Errors
Thermodynamics
Oligonucleotides
Communicable Diseases

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine

Cite this

Surpassing specificity limits of nucleic acid probes via cooperativity. / Satterfield, Brent C.; Bartosiewicz, Matt; West, Jay A A; Caplan, Michael.

In: Journal of Molecular Diagnostics, Vol. 12, No. 3, 01.05.2010, p. 359-367.

Research output: Contribution to journalArticle

Satterfield, BC, Bartosiewicz, M, West, JAA & Caplan, M 2010, 'Surpassing specificity limits of nucleic acid probes via cooperativity', Journal of Molecular Diagnostics, vol. 12, no. 3, pp. 359-367. https://doi.org/10.2353/jmoldx.2010.090056
Satterfield, Brent C. ; Bartosiewicz, Matt ; West, Jay A A ; Caplan, Michael. / Surpassing specificity limits of nucleic acid probes via cooperativity. In: Journal of Molecular Diagnostics. 2010 ; Vol. 12, No. 3. pp. 359-367.
@article{2834798d0a7b4587b5a373b2f6dccca9,
title = "Surpassing specificity limits of nucleic acid probes via cooperativity",
abstract = "The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.",
author = "Satterfield, {Brent C.} and Matt Bartosiewicz and West, {Jay A A} and Michael Caplan",
year = "2010",
month = "5",
day = "1",
doi = "10.2353/jmoldx.2010.090056",
language = "English (US)",
volume = "12",
pages = "359--367",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Association of Molecular Pathology",
number = "3",

}

TY - JOUR

T1 - Surpassing specificity limits of nucleic acid probes via cooperativity

AU - Satterfield, Brent C.

AU - Bartosiewicz, Matt

AU - West, Jay A A

AU - Caplan, Michael

PY - 2010/5/1

Y1 - 2010/5/1

N2 - The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

AB - The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

UR - http://www.scopus.com/inward/record.url?scp=77951743006&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77951743006&partnerID=8YFLogxK

U2 - 10.2353/jmoldx.2010.090056

DO - 10.2353/jmoldx.2010.090056

M3 - Article

VL - 12

SP - 359

EP - 367

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

IS - 3

ER -