Methods for Sequencing Individual Nucleic Acids Under Tension
The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.
1 - 81 . (canceled)
82 . A device comprising:
a substrate to immobilize a nucleic acid target, the nucleic acid target having a distal free end;
a fluidic subsystem to deliver at least one nucleotide to the substrate; and
a sensor to detect a change in a mechanical property of the nucleic acid target in response to incorporation of the nucleotide.
83 . The device of claim 82 , wherein the mechanical property includes a length associated with the nucleic acid target.
84 . The device of claim 82 , wherein the mechanical property includes a tension associated with the nucleic acid target.
85 . The device of claim 82 , wherein the sensor comprises a non-optical imaging system.
86 . The device of claim 82 , wherein the nucleotide comprises free of optically detected or fluorescent labels.
87 . The device of claim 82 , wherein the sensor comprises a non-contact sensor.
88 . The device of claim 87 , wherein the non-contact sensor comprises a CMOS device.
89 . The device of claim 87 , wherein the non-contact sensor comprises an ISFET array.
90 . The device of claim 82 , wherein a detectable moiety is coupled to the nucleic acid target.
91 . The device of claim 90 , wherein the detectable moiety is coupled to the distal free end of the nucleic acid target.
92 . The device of claim 90 , wherein the detectable moiety comprises a particle or bead.
93 . The device of claim 92 , wherein the particle or bead is magnetic.
94 . The device of claim 82 , wherein the fluidic subsystem comprises a flow cell providing a fluid flow having a substantially uniform direction across at least a portion of the flow cell.
95 . The device of claim 94 , wherein the flow subsystem provides at least partially laminar flow within the flow cell.
96 . The device of claim 82 , wherein the fluidic subsystem includes a flow resistor.
97 . The device of claim 82 , wherein the fluidic subsystem includes a pneumatic system for flowing of a reagent solution including the at least one nucleotide.
98 . The device of claim 82 , wherein the fluidic subsystem comprises a manifold configured to introduce a wash solution and reagent solutions.
99 . The device of claim 98 , wherein the manifold comprises a valve providing substantially zero unswept volume when closed.
100 . A device comprising:
a substrate to immobilize a nucleic acid target, the nucleic acid target having a distal free end;
a fluidic subsystem to deliver a nucleotide to the substrate, the fluidic system including a manifold, the manifold defining a flow path and including a wash solution valve, a first reagent solution valve, a second reagent solution valve, a third reagent solution valve and a fourth reagent solution valve, the first, second, third, and fourth reagent solution valves in fluid connection with the flow path downstream of the wash solution; and
a sensor to detect a change in a mechanical property of the nucleic acid target in response to incorporation of the nucleotide.
101 . The device of claim 100 , wherein the fluidic subsystem further comprises a flow cell associated with the substrate, the flow cell configured to provide substantially uniform directional flow across the substrate.
102 . The device of claim 101 , wherein the substantially uniform directional flow is substantially laminar.
103 . The device of claim 100 , wherein the fluidic subsystem further comprises a flow restrictor at an outlet of the flow cell.