Quantitative, highly multiplexed detection of nucleic acids
View Patent ↗This invention provides methods of detecting and quantifying target nucleic acids in samples in multiplexed single chamber reactions. Consumables incorporating chambers optimized to reduce signal background proximal to high efficiency arrays are provided, as well as methods of use. Devices and systems configured to use the consumables to practice the methods are a feature of the invention.
1. A method of detecting a target nucleic acid sequence in a sample, comprising:
performing an amplification reaction on the sample with a polymerase enzyme that possesses nuclease activity, in the presence of a reagent comprising first probes that comprise a first portion complementary to the target nucleic acid sequence and a second portion not complementary to the first target nucleic acid sequence, the second portion comprising a first quencher moiety coupled to the second portion at a first position, such that the second portion is cleaved from the first portion as a first probe fragment, when the target nucleic acid sequence is amplified;
hybridizing the first probe fragment to capture probes immobilized upon a substrate, wherein the capture probes comprise a fluorophore that is at least partially quenched by the first quencher moiety, the fluorophore coupled to a second position on the capture probes such that upon hybridization of the probe fragments to the capture probes, the fluorophore is at least partially quenched by the quencher; and
detecting the presence of the target sequence based upon the quenching of the fluorophore on the capture probes.
2. The method of claim 1 , wherein:
the amplification reagent comprises a plurality of different probes each different probe having a different first portions complementary to a different target nucleic acid sequences, and each different probe having a different second portions not complementary to a target nucleic acid sequences, the second portions of each different probe comprising a quencher moiety coupled to the first position, the second portion being cleaved as a probe fragments upon amplification of the plurality of target nucleic acid sequences; and
wherein the substrate comprises a plurality of different capture probe regions arrayed on the substrate, each capture probe region comprising capture probes complementary to a different second portion of the plurality of different probes, and each capture probe comprising a fluorophore quenched by the quencher moiety.
3. The method of claim 1 , wherein the first position comprises the 5′ end of the probe fragment, and the second position comprises the 3′ end of the capture probe.
4. 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.
5. The method of claim 1 , wherein the first probe fragment that hybridizes to the capture probes is less than about 30 nucleotides in length.
6. The method of claim 1 , wherein the first probe fragment that hybridizes to the capture probes is less than about 20 nucleotides in length.
7. The method of claim 1 , wherein the first probe fragment that hybridizes to the capture probes is less than about 15 nucleotides or less in length.
8. The method of claim 1 , wherein the target nucleic acid is amplified for at least 5 amplification cycles prior to said detecting.
9. The method of claim 1 , wherein the target nucleic acid is amplified in a plurality of amplification cycles prior to said detecting, wherein the target nucleic acid portion is additionally amplified after said detecting, in the presence of additional copies of the probe, with resulting released first probe fragments being subsequently hybridized to the array and detected, wherein detected signal intensity is correlated to a quantity of the target nucleic acid present in the sample.
10. The method of claim 1 , wherein a temperature of the hybridizing step is less than a temperature of the amplification reaction.
11. The method of claim 1 , comprising detecting local background for one or more regions of the array, and normalizing signal intensity measurements by correcting for said background.
12. The method of claim 2 , wherein the sample comprises a plurality of target nucleic acids.
13. The method of claim 2 , wherein the plurality of different capture probes are spatially separated on the substrate.
14. The method of claim 2 , wherein there are between about 5 and about 100 different capture probes, and between about 5 and about 100 corresponding probes in the amplification reaction, wherein up to 5 to 100 different signals can be detected based upon positioning of the signals in the array.
15. The method of claim 2 , wherein the capture probes are arrayed on the substrate at a density of about between about 350 fmoles/cm 2 and about 5,000 finoles/cm 2 or greater.
16. The method of claim 2 , wherein the capture probes are arrayed on the substrate at a density greater than 2000 firioles/cm 2 .
17. The method of claim 2 , wherein the plurality of different capture probes comprise the same fluorophore, and the plurality of different probe fragments comprise the same quencher moiety.
18. The method of claim 1 , wherein the amplifying step and the step of hybridizing the first probe fragment to the substrate are carried out at the same temperature.