Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion

A method for real-time fluorescent detection and quantification of nucleic acid amplification using a restriction endonuclease was developed. In this homogeneous system detection is mediated by a primer containing a reporter and quencher moiety at its 5' terminus separated by a short section of DNA encoding a restriction enzyme recognition sequence. In the single stranded form, the signal from the fluorescent reporter is quenched due to fluorescence resonance energy transfer. However, as the primer becomes incorporated into a double stranded amplicon, a restriction enzyme present in the reaction cleaves the DNA linking the reporter and quencher, allowing unrestricted fluorescence of the reporter. To test this system, a primer specific for the E6 gene of human papilloma virus (HPV) 16 was combined with the cleavable energy transfer label and used to amplify HPV16 positive DNA. In the presence of the thermally stable restriction enzyme BstNI, the reporter system was found to generate a fluorescent signal in proportion to the amount of template DNA. In addition to this direct format, the reporter primer was also used to monitor and quantify the amplification of other sequences. This was accomplished by using primers that contain a tag sequence complementary to the reporter oligonucleotide.