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Clinical Chemistry 49: 1599-1607, 2003; 10.1373/49.10.1599
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(Clinical Chemistry. 2003;49:1599-1607.)
© 2003 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System

Sha-Sha Wang2,1, Keith Thornton2,1, Andrew M. Kuhn1, James G. Nadeau1 and Tobin J. Hellyera,1

1 BD Diagnostic Systems, 54 Loveton Circle, Sparks, MD 21152.

aAuthor for correspondence. Fax 410-316-3690; e-mail Tobin_Hellyer{at}bd.com.

Background: The BD ProbeTecTM ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations.

Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human ß2-adrenergic receptor (ß2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing.

Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 µL of sample per reaction. All six ß2AR assays were able to accommodate >=20 µL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six ß2AR assays agreed with DNA sequencing results.

Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.




The following articles in journals at HighWire Press have cited this article:


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 Clin. Chem.Home page
T. Lucas, D. Losert, M. Allen, J. Halaschek-Wiener, B. Pratscher, C. Friedrich, M. Wolschek, G. Fuchsjager-Mayrl, L. Schmetterer, H. Pehamberger, et al.
Combination Allele-Specific Real-Time PCR for Differentiation of {beta}2-Adrenergic Receptor Coding Single-Nucleotide Polymorphisms
Clin. Chem., April 1, 2004; 50(4): 769 - 772.
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