Apparatus and method for fast digital detection
Methods and systems for sample target molecules are provided.
1. A method of detecting a target nucleic acid in multiple samples, the method comprising
contacting different samples comprising sample nucleic acids, optionally contained in vehicles, with different pluralities of solid supports to form different mixtures,
wherein the solid supports comprise a plurality of capture molecules that bind to the sample target nucleic acids or vehicles comprising the sample target nucleic acid, optionally wherein the plurality of capture molecules on a solid support are identical, and
wherein the solid supports contacted to different samples have a characteristic distinguishable from other solid supports contacted to different samples;
binding the capture molecules to the sample target nucleic acids or vehicles comprising the target nucleic acids, wherein the number of solid supports in the mixture is sufficiently high that at least some solid supports are not bound to target nucleic acids or vehicles comprising target nucleic acids;
combining the different mixtures to form a bulk mixture;
introducing the solid supports comprising bound sample target nucleic acids or vehicles into an array of wells, wherein the wells each can contain one but no more than one solid support;
detecting in each well (i) the presence or absence of the sample target nucleic acids and (ii) the characteristic of the solid support, wherein the detecting is performed with a scanner; and
determining with a computer processor the number of sample target nucleic acids per sample wherein the determining comprises determining the number of wells having detected sample target nucleic acids, wherein the sample identity is determined by the detected characteristic of the solid support.
2. The method of claim 1 , wherein after the binding and before or after the combining, washing unbound sample components from the solid supports.
3. The method of claim 1 , wherein the wells comprise affinity agents that bind the sample target nucleic acids or vehicles comprising sample target nucleic acids to the well.
4. The method of claim 1 , wherein after the introducing, covering the wells.
5. The method of claim 4 , further comprising amplifying the sample target nucleic acids.
6. The method of claim 5 , wherein after the sample target nucleic acids are amplified, the sample target nucleic acids are attached to the wells via an affinity agent linked to the wells.
7. The method of claim 1 , wherein the characteristic is the color of the solid support.
8. The method of claim 1 , wherein the capture molecules are capture oligonucleotides and the binding comprises hybridizing the capture oligonucleotides to the sample target nucleic acids and following the introducing and before the detecting, amplifying the sample target nucleic acids hybridized to the capture oligonucleotides, and wherein the detecting comprises detecting signal from a reagent in the wells that changes based on the presence or absence of amplified nucleic acids.
9. The method of claim 1 , wherein the sample target nucleic acids are RNA.
10. The method of claim 9 , wherein the RNA is reverse transcribed into cDNA.
11. The method of claim 10 , further comprising reverse transcribing the RNA before the introducing.
12. The method of claim 10 , further comprising reverse transcribing the RNA after the introducing.
13. The method of claim 1 , wherein the array of wells are of multiple different sizes or volumes.
14. The method of claim 13 , wherein the solid supports are beads having diameters no less than 10% smaller than the diameter of well openings.
15. The method of claim 1 , wherein the ratio of the diameter of the solid support to the diameter of the well is 1:1, or 1:2, or 1:3, or 1:4.
16. The method of claim 1 , wherein sets of wells are separated from each other such that individual samples can be deposited in each.
17. The method of claim 1 , wherein the solid supports contacted to the sample are of at least two types, wherein a first type comprises capture molecules that bind to a first sample target nucleic acid or a vehicle comprising the first sample target nucleic acid and a second type comprises capture molecules that bind to a second sample target nucleic acid or a vehicle comprising the second sample target nucleic acid.
18. The method of claim 1 , wherein the capture molecules are oligonucleotides complementary to the sample target nucleic acid and the binding comprises hybridization.
19. A method of detecting a target nucleic acid in multiple samples, the method comprising
contacting different samples comprising sample nucleic acids with different pluralities of solid supports to form different mixtures, wherein the solid supports comprise a plurality of capture molecules that bind to the sample target nucleic acid or a vehicle comprising the sample target nucleic acid, optionally wherein the plurality of capture molecules on a solid support are identical;
binding the capture molecules to the sample target nucleic acids, wherein the number of solid supports in the mixture is sufficiently high that at least some solid supports are not bound to target nucleic acids or vehicles comprising target nucleic acids;
introducing the solid supports comprising bound sample target nucleic acids or vehicles comprising sample target nucleic acids into an array of wells, wherein the wells each can contain no more than one solid support, wherein the position of wells receiving the sample beads is recorded such that wells corresponding to different samples is recorded;
detecting in each well (i) the presence or absence of the sample target nucleic acids and (ii) the position of the wells, wherein the detecting is performed with a scanner; and
determining with a computer processor the number of sample target nucleic acids per sample based in part on the number of wells comprising detected sample target nucleic acids, wherein the sample identity is determined by the position of the wells.
20. A method of detecting a target nucleic acid in multiple samples, the method comprising
contacting different samples comprising sample nucleic acids, optionally contained in vehicles, with different pluralities of solid supports to form different mixtures,
wherein the solid supports comprise a plurality of capture molecules that bind to the sample target nucleic acids or a vehicles comprising the sample target nucleic acid, optionally wherein the plurality of capture molecules on a solid support are identical, and
wherein the solid supports contacted to different samples have a characteristic distinguishable from other solid supports contacted to different samples;
binding the capture molecules to the sample target nucleic acids or vehicles comprising the target nucleic acids, wherein the number of solid supports in the mixture is sufficiently high that at least some solid supports are not bound to target nucleic acids or vehicles comprising target nucleic acids;
combining the different mixtures to form a bulk mixture;
introducing the solid supports comprising bound sample target nucleic acids or vehicles into an array of wells, wherein the wells can contain more than one solid support;
detecting in each well (i) the presence and intensity or absence of signal indicative of the sample target nucleic acids and (ii) the characteristic of the solid support, wherein the detecting is performed with a scanner; and
determining with a computer processor the number of sample target nucleic acids per sample wherein the determining comprises determining the number of wells having detected sample target nucleic acids and different signal intensities between wells together with the presence or absence of signal in each well is used to measure the concentration of target molecules in a sample, and wherein the sample identity is determined by the detected characteristic of the solid support.