Anti-primer quenching-based real-time PCR for simplex or multiplex DNA quantification and single-nucleotide polymorphism genotyping.

摘要: Nucleic acid amplification and detection plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics drug discovery. We present a new quantitative PCR method that allows versatile flexible nucleic target quantification. One the primers is modified by oligonucleotide “tail” fluorescently labeled at 5′ end. An complementary to this tail, carrying 3′-quencher (“anti-primer”), included along with two primers. Following primer extension, reaction temperature lowered such anti-primer hybridizes quenches fluorescence only free not double-stranded product, allowing real-time fluorescent quantification latter. This anti-primer-based (aQRT-PCR) simplex or multiplex single-nucleotide polymorphism genotyping samples widely differing quality (e.g., fresh formalin-fixed paraffin-embedded plasma-circulating DNA) provides practical alternative existing, more expensive approaches. The process aQRT-PCR takes 1.5–2 h.