Methods, kits and reaction mixtures for analyzing single-stranded nucleic acid sequences

摘要

Provided herein are fluorescence detection methods for nucleic acid sequences and to kits for performing such methods.

主权项

1. A homogeneous assay method for analyzing at least one single-stranded nucleic acid target sequence in a sample, comprising
a) providing a sample containing a nucleic acid comprising said at least one single-stranded nucleic acid target sequence and multiple detectably distinguishable probe sets, each set of the multiple detectably distinguishable probe sets comprising two probes: i) a probe labeled with a non-fluorescent quencher moiety that hybridizes to a region of one of said at least one single-stranded nucleic acid target sequence, and ii) a probe labeled with a fluorescent moiety that hybridizes to a different region of one of said at least one single-stranded nucleic acid target sequence adjacent to the region of said one of said at least one single-stranded nucleic acid target sequence that the probe labeled with the non-fluorescent quencher moiety hybridizes to,
wherein if said probe labeled with a fluorescent moiety hybridizes to said one of said at least one single-stranded nucleic acid target sequence in said sample in the absence of said probe labeled with a non-fluorescent quencher moiety, said fluorescent moiety emits a fluorescent signal, wherein, if both the probe labeled with a non-fluorescent quencher moiety and the probe labeled with a fluorescent moiety hybridize to said one of said at least one single-stranded nucleic acid target sequence, the non-fluorescent quencher moiety quenches the fluorescent signal from the fluorescent moiety; b) hybridizing said at least one single-stranded nucleic acid target sequence to the multiple detectably distinguishable probe sets so that the multiple detectably distinguishable probe sets hybridize to one of said at least one single-stranded nucleic acid target sequence and, after the hybridization, the multiple detectably distinguishable probe sets are arranged adjacent to each other on one of said at least one single-stranded nucleic acid target sequence, and there is no space between each of the multiple detectably distinguishable probe sets; and c) analyzing the hybridization of the multiple detectably distinguishable probe sets to said one of said at least one single-stranded nucleic acid target sequence by assessing a fluorescent signal produced in step b) as a function of temperature.