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 sequencing a nucleic acid, the method comprising:
immobilizing a nucleic acid target including a plurality of tandem repeat nucleic acid sequences;
performing a polymerase-based extension to incorporate at least one known nucleotide into the nucleic acid target;
detecting the incorporation of the at least one known nucleotide in a non-fluorescence based manner; and
determining at least one nucleotide within the sequence of the nucleic acid target based on the identity of the incorporated at least one known nucleotide.
83 . The method of claim 82 , wherein performing the detection comprises performing detection in the absence of enzymes other than a polymerase.
84 . The method of claim 82 , wherein performing the detection comprises performing detection without detecting sequencing byproducts.
85 . The method of claim 82 , wherein the at least one known nucleotide are un-labeled or unmodified.
86 . The method of claim 82 , wherein immobilizing the nucleic acid target includes coupling the nucleic acid target to a linker or primer secured to a substrate.
87 . The method of claim 82 , wherein performing detection comprises determining a mechanical property of the extended nucleic acid target.
88 . The method of claim 87 , wherein the mechanical property comprises a length associated with the nucleic acid target which changes under tension when a nucleotide is incorporated.
89 . The method of claim 88 , wherein the tension is in a range of approximately 1 pN to 12 pN.
90 . The method of claim 88 , wherein a flow-based force induces tension in the nucleic acid target.
91 . The method of claim 88 , wherein a magnetic force induces tension in the nucleic acid target.
92 . The method of claim 88 , wherein an electrical force induces tension in the nucleic acid target.
93 . The method of claim 88 , wherein at least two different forces induce tension in the nucleic acid target.
94 . The method of claim 82 , wherein the nucleic acid target includes a detectable moiety coupled to the nucleic acid target.
95 . The method of claim 94 , wherein the detectable moiety includes a particle or bead.
96 . The method of claim 95 , wherein the particle or bead is magnetic.
97 . A method for nucleic acid sequencing, the method comprising:
immobilizing a nucleic acid target including a plurality of tandem repeat nucleic acid sequences;
performing a polymerase-based extension to incorporate at least one known nucleotide into the nucleic acid target;
detecting the incorporation of the at least one known nucleotide based on a change in a mechanical property of the extended nucleic acid target; and
determining at least one nucleotide within the sequence of the nucleic acid target based on the identity of the incorporated at least one known nucleotide.
98 . The method of claim 97 , wherein the mechanical property is associated with the length of the nucleic acid target that changes under tension when a nucleotide is incorporated.
99 . The method of claim 98 , wherein the tension is in a range of approximately 1 pN to 12 pN.
100 . The method of claim 98 , wherein a flow-based force induces tension in the nucleic acid target.
101 . The method of claim 98 , wherein a magnetic force induces tension in the nucleic acid target.
102 . The method of claim 98 , wherein an electrical force induces tension in the nucleic acid target.
103 . The method of claim 98 , wherein at least two different forces induce tension in the nucleic acid target.
104 . A method for sequencing a nucleic acid sequence, the method comprising:
immobilizing a nucleic acid target including a plurality of tandem repeat nucleic acid sequences, the nucleic acid target including a detectable moiety coupled proximal to a free end of the nucleic acid target;
performing a polymerase-based extension to incorporate a nucleotide onto the nucleic acid target; and
determining whether the nucleotide is incorporated based on a change in a mechanical property of the extended nucleic acid target indicated by a change in position of the detectable moiety.
105 . A method for nucleic acid sequencing comprising:
coupling a plurality of tandemly oriented nucleic acids on a solid support surface;
performing a sequencing reaction using the tandemly oriented nucleic acids as templates, and
detecting incorporation of nucleotides in the absence of fluorescence, wherein the plurality of template nucleic acids are bound to a separate region of the solid support surface that is contiguous and in fluid communication with other regions on the solid support surface.