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 method for analyzing a nucleic acid target, the method comprising:
immobilizing a primer to a substrate, the primer including a region complementary to a nucleic acid template;
hybridizing the nucleic acid template with the primer;
synthesizing a nucleic acid target complementary to the nucleic acid template by rolling circle amplification to provide a plurality of copies of tandemly-arranged nucleic acid template; and
interrogating the nucleic acid target using a non-fluorescence based detection method.
83 . The method of claim 82 wherein, the non-fluorescence based detection method is based on a change in a mechanical property of the nucleic acid target.
84 . The method of claim 83 , wherein the mechanical property of the nucleic acid target is associated with the length of the nucleic acid target that changes during the interrogation.
85 . The method of claim 83 , wherein a flow-based force induces a change in the mechanical property in the nucleic acid target.
86 . The method of claim 83 , wherein a magnetic force induces a change in the mechanical property in the nucleic acid target.
87 . The method of claim 83 , wherein an electrical force induces a change in the mechanical property in the nucleic acid target.
88 . The method of claim 83 , wherein at least two different forces induces a change in the mechanical property in the nucleic acid target.
89 . The method of claim 82 , wherein immobilizing comprises non-covalently coupling the primer to the substrate.
90 . The method of claim 89 , wherein non-covalently coupling includes coupling to a linker.
91 . The method of claim 90 , wherein the linker comprises polyethylene glycol.
92 . The method of claim 82 , wherein immobilizing comprises coupling a biotin-containing primer to the substrate having streptavidin disposed thereon.
93 . The method of claim 82 , wherein immobilizing includes covalently coupling.
94 . A method for sequencing a nucleic acid target, the method comprising:
ligating nucleic acid template to form a circular nucleic acid template;
immobilizing a primer to a substrate, the primer including a region complementary to the circular nucleic acid template;
coupling the circular nucleic acid template to the primer;
synthesizing a nucleic acid target complementary to at least a portion of the circular nucleic acid template, the synthesized nucleic acid target having a plurality of copies of at least a portion of the circular nucleic acid template arranged in tandem; and
performing a sequencing reaction which includes a non-fluorescent detection mechanism to identify at least a portion of the nucleic acid target sequence.
95 . The method of claim 94 , wherein immobilizing includes non-covalently coupling the primer to the substrate.
96 . The method of claim 95 , wherein non-covalently coupling includes coupling to a linker.
97 . The method of claim 96 , wherein the linker comprises polyethylene glycol.
98 . The method of claim 94 , wherein immobilizing includes coupling a biotin-containing primer to a substrate having streptavidin disposed thereon.
99 . The method of claim 94 , wherein immobilizing comprises covalently bonding.
100 . The method of claim 94 wherein, the non-fluorescence based detection mechanism is based on a change in a mechanical property of the nucleic acid target.
101 . The method of claim 100 , wherein the mechanical property of the nucleic acid target is associated with the length of the nucleic acid target that changes during the sequencing reaction.
102 . The method of claim 100 , wherein a flow-based force induces a change in the mechanical property in the nucleic acid target.
103 . The method of claim 100 , wherein a magnetic force induces a change in the mechanical property in the nucleic acid target.
104 . The method of claim 100 , wherein an electrical force induces a change in the mechanical property in the nucleic acid target.
105 . The method of claim 100 , wherein at least two different forces induces a change in the mechanical property in the nucleic acid target.