SNP Genotyping Using Single-Tube Fluorescent Bidirectional PCR

• Published in: BioTechniques Vol. 33; no. 1; pp. 80 - 90
• Main Authors: Waterfall, Christy M, Cobb, Benjamin D
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.07.2002; Eaton
• Subjects: Anemia, Sickle Cell - genetics; Biological and medical sciences; Biotechnology; DNA Mutational Analysis - instrumentation; DNA Mutational Analysis - methods; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic Markers; Genetic technics; Genome, Human; Genotype; Humans; In vitro gene amplification. Pcr technique; Methods. Procedures. Technologies; Nucleic Acid Heteroduplexes - chemistry; Nucleic Acid Heteroduplexes - metabolism; Organic Chemicals; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Polymorphism, Single Nucleotide - genetics; Reproducibility of Results; Restriction Mapping - methods; Sensitivity and Specificity; Single-Blind Method; Templates, Genetic; Polymerase chain reaction; Fluorescence; Genotype; Tube
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/02331st05

SNP genotyping is a well-populated field with a large number of assay formats offering accurate allelic discrimination. However, there remains a discord between the ultimate goal of rapid, inexpensive assays that do not require complex design considerations and involved optimization strategies. We describe the first integration of bidirectional alleles-pecific amplification, SYBR Green®I, and rapid-cycle PCR to provide a homogeneous SNP-typing assay. Wild-type, mutant, and heterozygous alleles were easily discriminated in a single tube using melt curve profiling of PCR products alone. We demonstrate the effectiveness and reliability of this assay with a blinded trial using clinical samples from individuals with sickle cell anemia, sickle cell trait, or unaffected individuals. The tests were completed in less than 30 min without expensive fluorogenic probes, prohibiting design rules, or lengthy downstream processing for product analysis.