Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes

Over the past 5 years, there has been substantial progress in sequencing the human genome and identifying clinically significant genes (1). Genes that are clinically significant are diagnostic or prognostic for disease and/or helpful in guiding treatment. Unknown gene mutations, resulting from germline or somatic DNA alterations, are initially defined by direct sequencing. Other methods that detect specific mutations can then be used for higher throughput. Recently developed instrumentation and techniques for genotyping combine PCR and fluorescent hybridization probes for homogeneous amplification and product analysis within 1 h (2)(3)(4)(5). The target is amplified from genomic DNA by rapid-cycle PCR (6) with all the reagents needed for genotyping present from the beginning of the reaction. After 15–20 min, PCR is completed and the instrument automatically begins a melting curve protocol. Fluorescence is acquired continuously as the reaction is slowly heated and genotypes are identified by their characteristic melting curves. Because amplification and genotyping occur in the same instrument in a closed tube format, there is no concern of contamination by previously amplified product. Hybridization probes are oligonucleotides that are singly labeled with a donor or acceptor fluorophore. During probe/target hybridization, these fluorophores are brought into close proximity and fluorescence resonance energy transfer occurs. Two hybridization probe schemes for fluorescent resonance energy transfer have been developed (3)(5). One method uses a 3′-labeled hybridization probe designed to anneal to a PCR strand …