COMBINATION OF DSDNA BINDING DYE AND PROBES FOR CHARACTERIZATION OF SSDNA SEQUENCES

摘要

This invention includes methods for analyzing single-stranded nucleic acid sequences, either RNA sequences or DNA sequences (ssDNA) utilizing dyes that fluoresce when associated with double strands, so-called DNA binding dyes or dsDNA-dyes. Methods according to this invention utilize a dsDNA-dye in combination with one or more hybridization probes that hybridize to a target nucleic acid sequence and that are labeled with a non-fluorescent quencher moiety, for example, a Black Hole quencher.

主权项

1. A homogeneous method for analyzing the nucleic acid content of at least one nucleic acid target sequence contained in copies of at least one nucleic acid target strand as a function of temperature, comprising
a) providing a sample containing copies of said at least one nucleic acid target strand in single-stranded form, said copies having a melting temperature relative to their complementary nucleic acid strands; b) contacting said single-stranded copies with a double strand DNA binding dye (dsDNA-dye) and a probe set that includes at least one hybridization probe that is complementary to said at least one target sequence and is labeled with at least one non-fluorescent moiety that is a quencher for the dsDNA-dye; c) subjecting the sample, at multiple temperatures that are below the melting temperature of said copies, to excitation at a wavelength appropriate for stimulating the dye and detecting emission at a wavelength appropriate for detecting emission from the dsDNA-dye; and d) comparing the detected emissions to corresponding emissions obtained from at least one target sequence whose sequence or nature is known.