Fluorescent stokes shift probes for polynucleotide hybridization assays

Claim 1

The spectroscopy method for detecting a target single-strand polynucleotide sequence in a polynucleotide sample in which either (i) a fluorescent polynucleotide single probe sequence complementary to said target sequence is hybridized thereto, or (ii) a plurality of probes of such sequences complementary to sequentially adjacent portions of said target sequences are hybridized thereto, wherein the improvement comprises having present in said single proble or in said plurality of probes at least one pair of fluorescent moieties connected by linker arms to nucleic acid base units, said fluorescent moieties comprising respectively donor and acceptor moieties selected so that the emission spectrum of the donor moiety overlaps the excitation spectrum of the acceptor moiety to permit non-radiative energy transfer with efficient fluorescent emission by the acceptor fluorophores, the wavelength maximum of the emission spectrum of the acceptor moiety being at least 100nm greater than the wavelength maximum of the excitation spectrum of the donor moiety, said linker arms having lengths of 4 to 30 Angstroms, the donor and acceptor moieties being connected to non-contiguous base units in said single probe or to base units in said plural probes other than 3' and 5' end units thereof, when said single probe or said plural probes are hybridized to the target sample the base units to which said donor and acceptor moieties are connected being paired through hybridization to base units of said target sequence which are separated by 2 to 7 intervening nucleotide base units of the target sequence.

Claim 10

The method of claims 6, 7, or 8 in which said donor moiety is fluorescein and said acceptor moiety is Texas Red.

Claim 11

The method of claims 1 or 6 in which said donor and acceptor moieties are on a single probe and are connected to base units thereof other than the 3' and 5' end units and which are separated by 4 to 6 intervening base units.

Claim 12

The method of claims 1 or 6 in which said moieties are on a pair of probes respectively having 3' and 5' ends hybridizing to adjacent base units of the target polynucleotide and when so hybridized the donor and acceptor moieties being separated by 4 to 6 intervening base units.