PASSIVE INTERNAL REFERENCES FOR THE DETECTION OF NUCLEIC ACID AMPLIFICATION PRODUCTS

The invention relates to passive internal references for use in quantitating the formation of amplification products in a nucleic amplification reaction. The internal amplification reference molecules of the invention comprise a first and second fluorophore joined together through a backbone connector. The first and second fluorophores are joined on the backbone in a configuration that permits the energy transfer from the first fluorophore to the second fluorophore. The backbone connector is selected so as not to bind to the target nucleic acid sequence under nucleic acid amplification conditions. Preferably, the backbone connector is a polynucleotide. Another aspect of the invention is to provide passive internal reference molecule containing reagent compositions for use in nucleic acid amplification reactions. The compositions comprise the internal amplification reference molecule of the invention and a nucleic acid amplification reaction buffer. The reagent compositions, optionally, include additional components required for nucleic acid amplification reactions. The invention also provides improved methods of measuring the amount of amplification product in nucleic acid amplification reactions employing fluorescer-quencher probe assays, including methods for the real-time measurement of amplification product formation. The methods comprise the step of adding the internal reference molecule of the invention to the amplification reaction mixture. Fluorescence of the second fluorophore on the internal reference may then be measured and used to calculate changes in fluorescence of the fluorophore on a fluorescer-quencher probe.