SNP genotyping using single-tube fluorescent bidirectional PCR.
SNP genotyping is a well-populatedfield 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 allele-specific 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.
Molecular Sensing plc, Melksham, Wiltshire, UK. christy.waterfall@molecular-sensing.com
Christy M
CM
Waterfall
Benjamin D
BD
Cobb
D000755:Anemia, Sickle Cell / Q000235:genetics*; D052160:Benzothiazoles; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D003959:Diamines; D005456:Fluorescent Dyes; D005819:Genetic Markers; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011804:Quinolines; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D013698:Templates, Genetic
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D005819:Genetic Markers; D009692:Nucleic Acid Heteroduplexes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I