Sequencing Nucleic Acid Polymers with Electron Microscopy
This invention relates to using an electron microscope to sequence by direct inspection of labeled, stretched DNA. This method will have higher accuracy, lower cost, and longer read length than current DNA sequencing methods.
1 - 93 . (canceled)
94 . A system for determining a sequence of a nucleic acid, the system comprising:
(a) a data analysis module configured to:
(i) receive electron microscopy (EM) data obtained by imaging a contrast agent labeled nucleic acid with an electron microscope; and
(ii) determine a sequence of the nucleic acid from the EM data; and
(b) an output configured to provide the sequence of the nucleic acid to a user;
wherein the system is configured to provide the sequence at a rate of 1,000 bases per second or greater.
95 . The system according to claim 94 , wherein the sequence comprises 20 or more consecutive bases.
96 . The system according to claim 95 , wherein the sequence comprises 50 or more consecutive bases.
97 . The system according to claim 96 , wherein the system further comprises an electron microscope.
98 . The system according to claim 97 , wherein the electron microscope is a transmission electron microscope.
99 . The system according to claim 98 , wherein the transmission electron microscope is configured to obtain image data at a rate of 10,000 or more bases per second.
100 . The system according to claim 99 , wherein the system further comprises an imaging substrate configured to display a nucleic acid having consistent base-to-base spacing on a surface thereof.
101 . The system according to claim 100 , wherein the system is configured to provide a nucleic acid having consistent base-to-base spacing on the imaging substrate.
102 . The system according to claim 101 , wherein the system comprises a nucleic acid binding tool.
103 . The system according to claim 102 , wherein the nucleic acid binding tool is a needle.
104 . The system according to claim 103 , wherein the needle is disposed on a nanopositioner driven support.
105 . The system according to claim 104 , wherein the nanopositioner support comprises two or more needles.
106 . The system according to claim 104 , wherein the system is configured to dip the needle into and out of a nucleic acid fluid at a rate ranging from 1 nm/hr to 10 m/s.
107 . The system according to claim 105 , wherein the system is configured dip the needle into a fluid at a distance ranging from 1 Å to 20 μm.
108 . The system according to claim 106 , wherein the needle comprises a tip member having a radius of curvature less than about 200 nm functionalized with a compound that binds to a nucleic acid.
109 . The system according to claim 102 , wherein the system is configured position a nucleic acid on the imaging substrate with the nucleic acid binding tool by using a molecular threading protocol.
110 . The system according to claim 109 , wherein the molecular threading protocol is a shelf-threading protocol.
111 . The system according to claim 109 , wherein the molecular threading protocol is a gap-threading protocol.
112 . The system according to claim 109 , wherein the system is configured to transfer a nucleic acid from an initial substrate to the imaging substrate.
113 . The system according to claim 100 , wherein the system further comprises a nucleic acid having consistent base-to-base spacing on the imaging substrate.
114 . The system according to claim 106 , wherein the system further comprises a nucleic acid fluid comprising a nucleic acid to be sequenced.
115 . An electron microscope imaging substrate comprising a nucleic acid having consistent base-to-base spacing over a length of 1000 base pairs on a surface thereof.
116 . The electron microscope imaging substrate according to claim 115 , wherein the nucleic acid has a linear configuration.
117 . The electron microscope imaging substrate according to claim 116 , wherein the substrate comprises two or more parallel linear nucleic acids each having consistent base-to-base spacing over a length of 1000 base pairs.
118 . A needle comprising a tip member having a radius of curvature less than about 200 nm that has been functionalized with a nucleic acid binding compound.
119 . The needle according to claim 118 , wherein the needle further comprises a coating of a second compound that does not have an affinity for binding to a nucleic acid.
120 . The needle according to claim 119 , wherein the needle is disposed on a nanopositioner-driven support.
121 . The needle according to claim 120 , wherein the nanopositioner-driven support comprises two or more needles.