ABSTRACT
Strand displacement amplification is an isothermal process that permits 10(10)-fold amplification of a DNA target sequence in as little as 15 min. In the form of the BD ProbeTec ET System, strand displacement amplification was the first nucleic acid amplification technology to be coupled with real-time homogeneous fluorescence-based detection for routine application in the clinical laboratory. The isothermal nature of the reaction process offers distinct advantages with regard to the cost and simplicity of instrumentation, while a universal detection format permits the use of the same fluorescent detector probes across multiple analytes. This has important potential in the field of genetic analysis, in which disease predisposition and therapeutic efficacy are frequently determined by multiple nucleic acid markers.
Subject(s)
DNA/chemistry , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Base Sequence , DNA/metabolism , Humans , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , Sensitivity and SpecificityABSTRACT
BACKGROUND: The BD ProbeTec 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 beta(2)-adrenergic receptor (beta(2)AR) 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 micro L of sample per reaction. All six beta(2)AR assays were able to accommodate >/==" BORDER="0">20 micro L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six beta(2)AR 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.
