Method for detection of nucleic acid using total internal reflectance

Claim 1

A method of detecting amplified target nucleic acid using total internal reflection, comprising the steps providing a closed static-volumetric reaction vessel having disposed therein (a) reaction sample, containing the target nucleic acid, (b) a total internal reflection (TIR) element, (c) a plurality of members of at least one initiator sequence set and reagents for producing amplification of target nucleic acid present in the reaction sample, (d) label means which is coupled to a fluorophore, and (e) capture means for bringing said fluorophore within the penetration depth of said element, wherein said label means or said capture means is specific for said target nucleic acid; producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth; reacting the reaction sample, the members of said initiator sequence set and amplification reagents under conditions sufficient to amplify target nucleic acid present in the reaction sample to produce amplification products wherein said amplification products are labeled with said label means during amplification or said amplification products are labeled with said labels means by contacting said amplification products with a specific binding member attached to a label; capturing said label amplification products within the penetration depth as a function of the presence or amount of target nucleic acid; and detecting within the TIR element a change in fluorescence as a measure of target nucleic acid to thereby amplify and detect said target sequence in said closed static-volumetric reaction vessel.

Claim 2

A method of detecting amplified target nucleic acid using total internal reflection, comprising the steps providing a closed static-volumetric reaction vessel having disposed therein (a) reaction sample, containing the target nucleic acid, (b) a total internal reflection (TIR) element, (c) a plurality of members of at least one initiator sequence set and reagents for producing amplification of target nucleic acid present in the reaction sample, (d) label means which is coupled to a fluorophore, and (e) capture means for bringing said fluorophore within the penetration depth of said element, wherein said label means or said capture means is specific for said target nucleic acid; producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth; reacting the reaction sample, the members of said initiator sequence set and amplification reagents under conditions sufficient to amplify target nucleic acid present in the reaction sample to produce amplification products wherein said amplification products are labeled with said label means during amplification or said amplification products are labeled with said labels means by contacting said amplification products with a specific binding member attached to a label; capturing said label amplification products within the penetration depth as a function of the presence or amount of target nucleic acid; and detecting within the TIR element a change in fluorescence as a measure of target nucleic acid to thereby amplify and detect said target sequence in said closed static-volumetric reaction vessel.

Claim 14

A method of detecting target nucleic acid in a reaction vessel using total internal reflection, comprising the steps of: providing a closed static-volumetric reaction vessel having disposed therein (a) a reaction sample, (b) a total internal reflection (TIR) element, (c) a plurality of members of an initiator sequence, each sequence comprising a capture segment linked to a label segment by a scissile linkage, wherein said initiator includes a portion of nucleotide sequences which are capable of hybridizing with the target nucleic acid, and wherein said label segment is coupled to a fluorophore, and (d) means for cleaving the scissile linkage when target is hybridized to the initiator; producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth, and detecting in the TIR element the fluorescence resulting from the linked fluorophore present within the penetration depth; reacting the reaction sample, initiator sequences and means for cleaving under conditions sufficient to (a) hybridize target nucleic acid present in the reaction sample to the initiator sequences, and (b), in the presence of target, cleave the scissile linkage, thereby freeing the fluorophore from the penetration depth; and detecting in the TIR element a change in fluorescence as a measure of target nucleic acid to thereby react and detect said target nucleic acid in said closed static-volumetric reaction vessel.

Claim 15

A method of detecting target nucleic acid in a reaction vessel using total internal reflection, comprising the steps of: providing a closed static-volumetric reaction vessel having disposed therein (a) a reaction sample, (b) a total internal reflection (TIR) element, (c) a plurality of members of an initiator sequence, each sequence comprising a capture segment linked to a label segment by a scissile linkage, wherein said initiator includes a portion of nucleotide sequences which are capable of hybridizing with the target nucleic acid, and wherein said label segment is coupled to a fluorophore, and (d) means for cleaving the scissile linkage when target is hybridized to the initiator; producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth, and detecting in the TIR element the fluorescence resulting from the linked fluorophore present within the penetration depth; reacting the reaction sample, initiator sequences and means for cleaving under conditions sufficient to (a) hybridize target nucleic acid present in the reaction sample to the initiator sequences, and (b), in the presence of target, cleave the scissile linkage, thereby freeing the fluorophore from the penetration depth; and detecting in the TIR element a change in fluorescence as a measure of target nucleic acid to thereby react and detect said target nucleic acid in said closed static-volumetric reaction vessel.