JP2014533502A5 - - Google Patents

Claims (18)

A method for detecting a target nucleic acid sequence in a sample, comprising:
Using a polymerase enzyme having nuclease activity in the presence of a reagent comprising a first probe comprising a first portion complementary to a target nucleic acid sequence and a second portion not complementary to the first target nucleic acid sequence, to a sample Performing an amplification reaction, wherein the second portion includes a first quencher portion that is coupled to the second portion at the first portion so that the second portion is amplified when the target nucleic acid sequence is amplified; Cleaved from the first part as a first probe fragment;
Hybridizing a first probe to a capture probe immobilized on a substrate, wherein the capture probe comprises a fluorophore that is at least partially quenched by a first quencher moiety, The fluorophore molecule is at least partially quenched by the quencher when it binds to a second portion on the capture probe so that the probe fragment hybridizes to the capture probe;
Detecting the presence of a target sequence based on quenching of the fluorophore on the capture probe. The amplification reagent includes a plurality of different probes, each different probe having a different first portion complementary to a different target nucleic acid sequence, and each different probe having a different second portion that is not complementary to the target nucleic acid sequence. The second portion of each different probe includes a quencher portion attached to the first portion, the second portion being cleaved as a probe fragment upon amplification of a plurality of target nucleic acid sequences;
Here, the substrate includes a plurality of different capture probe regions aligned with the substrate, each capture probe region includes a capture probe that is complementary to a different second portion of the plurality of different probes, and each capture probe is The method of claim 1, comprising a fluorescent dye molecule that is quenched by a quencher moiety. 'Includes an end, the second portion 3 of the capture probe' 5 of the first portion probe fragment comprising the end, the method according to claim 1. 3. The method of claim 2 , wherein each capture probe region comprises a non-rate limiting number of capture nucleic acids that hybridize to the probe fragment.   The method of claim 1, wherein the first probe fragment that hybridizes to the capture probe is less than about 30 nucleotides in length.   The method of claim 1, wherein the first probe fragment that hybridizes to the capture probe is less than about 20 nucleotides in length.   2. The method of claim 1, wherein the first probe fragment that hybridizes to the capture probe is less than about 15 nucleotides in length.   2. The method of claim 1, wherein the target nucleic acid is amplified for at least 5 amplification cycles prior to detection.   The target nucleic acid is amplified in amplification during multiple amplification cycles prior to detection, wherein the target nucleic acid portion is further amplified after detection in the presence of additional copies of the probe, followed by the resulting release sequence. The method of claim 1, wherein one probe fragment is hybridized to the array and detected, wherein the detected signal intensity correlates with the amount of target nucleic acid present in the sample.   The method of claim 1, wherein the hybridization temperature does not exceed the temperature of the amplification reaction.   The method of claim 1, comprising normalizing signal intensity measurements by detecting a local background for one or more regions of the array and correcting for the background.   The method of claim 2, wherein the sample comprises a plurality of target nucleic acids.   The method of claim 2, wherein a plurality of different capture probes are spatially separated on the substrate.   About 5 to about 10 different capture probes and about 5 to about 100 corresponding probes are present in the amplification reaction, where up to 5-100 different signals are based on the positioning of the signal in the array. The method of claim 2, which can be determined. The method of claim 2, wherein the capture probes are aligned on the substrate at a density from about 350 fmol / cm ² to greater than about 5,000 fmol / cm ² . The method of claim 2, wherein the capture probes are aligned on the substrate at a density greater than 2000 fmol / cm ² . A plurality of different capture probes comprise the same fluorescent dye component child, comprising a plurality of different probes fragments of the same quencher moiety, A method according to claim 2.   The method of claim 1, wherein the amplifying step and the step of hybridizing the first probe fragment to the substrate are performed at the same temperature.